Prodrugs built as multiple self-elimination-release spacers

ABSTRACT

This invention concerns multiple release spacers and spacer systems, which release multiple leaving groups following a single activation. It concerns compounds comprising a specifier linked to two or more of the same or different leaving groups (L in the figure) via a self-eliminating multiple release spacer or spacer system, which compounds upon a single activation step, in particular removal or transformation of the specifier, release at least two leaving groups.

FIELD OF THE INVENTION

This invention is directed to compounds or prodrugs comprising amultiple release spacer or spacer system. A multiple release spacer isdefined as a self-elimination spacer that releases multiple leavinggroups upon a single activation event. One or more (generations) ofthese multiple release spacers can be used for example to obtainconjugates or prodrugs which are activated by a single activation stepat the preferred site of action in order to selectively deliver multipletherapeutic or diagnostic parent moieties to target cells or to a targetsite. In this invention preferably target cells are tumor cells.

BACKGROUND OF THE INVENTION

Lack of selectivity of chemotherapeutic agents is a major problem incancer treatment. Because highly toxic compounds are used in cancerchemotherapy, it is typically associated with severe side effects. Drugconcentrations that would completely eradicate the tumor cannot bereached because of dose-limiting side effects such as gastrointestinaltract and bone marrow toxicity. In addition, tumors can developresistance against anticancer agents after prolonged treatment. Inmodern drug development, targeting of cytotoxic drugs to the tumor sitecan be considered one of the primary goals.

A promising approach to obtain selectivity for tumor cells or tumortissue is to exploit the existence of tumor-associated enzymes. Arelatively high level of tumor-specific enzyme can convert apharmacologically inactive prodrug to the corresponding active parentdrug in the vicinity of or inside the tumor. Via this concept a highconcentration of toxic anticancer agent can be generated at the tumorsite. All tumor cells may be killed if the dose is sufficiently high,which may decrease development of drug resistant tumor cells.

There are several enzymes that are present at elevated levels in certaintumor tissues. One example is the enzyme β-glucuronidase, which isliberated from certain necrotic tumor areas. Furthermore, severalproteolytic enzymes have been shown to be associated with tumor invasionand metastasis. Several proteases, like for example the cathepsins andproteases from the urokinase-type plasminogen activator (u-PA) systemare all involved in tumor metastasis. The serine protease plasmin playsa key role in tumor invasion and metastasis. The proteolytically activeform of plasmin is formed from its inactive pro-enzyme form plasminogenby u-PA. The tumor-associated presence of plasmin can be exploited fortargeting of plasmin-cleavable conjugates or prodrugs.

An enzyme can also be transported to the vicinity of or inside targetcells or target tissue via antibody-directed enzyme prodrug therapy(ADEPT)¹, polymer-directed enzyme prodrug therapy (PDEPT) ormacromolecular-directed enzyme prodrug therapy (MDEPT)², virus-directedenzyme prodrug therapy (VDEPT)³ or gene-directed enzyme prodrug therapy(GDEPT)⁴.

The technology of this invention relates to novel spacers (linkers) orspacer systems (linker systems) that can be inserted between a specifier(unit that can be cleaved or transformed) and leaving groups (forexample parent drugs or detectable molecules). Furthermore, theinvention relates to prodrugs and (bio)conjugates comprising aspecifier, said novel spacers or spacer systems and multiple leavinggroups, and to bifunctional linker systems comprising a (protected)specifier containing a reactive moiety that enables coupling to atargeting moiety on one side of the linker (system) and reactivemoieties that enable coupling to multiple leaving groups (for exampleparent drugs or detectable molecules) on the other side of the linker(system). A great number of anticancer conjugates and prodiligs thathave been developed in the past contain a self-eliminating connector orlinker, also called self-elimination spacer. This spacer is incorporatedbetween the specifier and the drug in order to facilitate enzymaticcleavage and so enhance the kinetics of drug release (as shown in FIG.1). The specifier (which for example can be an oligopeptide substratefor a protease or for example a β-glucuronide substrate forβ-glucuronidase) must be site-specifically removed or transformed,followed by a spontaneous spacer elimination to release the cytotoxicparent drug. Up to now self-elimination spacers have been implementedthat release one drug molecule upon prodrug activation and subsequentspacer elimination. When the prodrugs and (bio-)conjugates containingmultiple drug moieties are considered that have been reported thus far,an independent cleavage was necessary for each drug molecule to bereleased.

WO 98/13059 is a relevant disclosure describing a prodrug comprising anamino-terminal capped peptide covalently linked to a therapeutic drugthrough a self-eliminating spacer. In particular this document describesthe use of p-aminobenzyl-oxycarbonyl (PABC) as self-elimninating spacer.The PABC electronic cascade spacer was already known from for instanceCarl et al., J. Med. Chem., 1981, vol. 24, 479-480. Specifically theanticancer drugs doxorubicin, mitomycin C, paclitaxel and camptothecincoupled to PABC are described. A second self-eliminating spacer that isdescribed is p-amino-bis(hydroxymethyl)styrene (BHMS), having thestructure p—NH—Ph—CH═C(CH₂O—)₂, including the carbonyl groups thestructure is p—NH—Ph—CH═C(CH₂OCO—)₂. It is noted that this spacer isdescribed in this disclosure as a bis-carbamate, which teaches that twodrug molecules are linked to this spacer via an amine functionality ofthe drug. The only drug moiety that is disclosed that is actuallycoupled to the BHMS spacer is doxorubicin. Doxorubicin is coupled viaits sugar amino group resulting in a carbamate linkage between spacerand drug. Further it is stated that the spacer can bind two drugmoieties. However, the document is silent on how many drug molecules areactually released.

Other systems that are loaded with multiple covalently bound bioactivemolecules as end groups have been reported. Examples are systems thatrelease doxorubicin after acidolysis of each hydrazone linker⁵, starlikeHPMA copolymers containing doxorubicin⁶, or multi-loaded poly(ethyleneglycol) prodrugs⁷. A doxorubicin-containing starlike HPMA copolymer withan antibody as the core has also been reported⁸. A number of recentpublications have described the use of branched linkers in combinationwith antibody-containing prodrugs or bioconjugates with the aim ofincreasing the number of drugs bound per antibody⁹. However, to ourknowledge, in each multi-loaded prodrug system reported so far, eachsingle end group needs to be independently cleaved in order to releaseall end groups.

Thus there is a need for improved prodrugs or (bio-)conjugates in termsof (efficiency of) release of sufficient amounts of active drug inrelation to the activation that is required at a desired site of action.In many cases, in prodrugs or (bio)conjugates, it is desirable toincrease drug loading per targeting unit, in order to improve theefficacy of targeted compounds.

SUMMARY OF THE INVENTION

The present invention fulfills the above mentioned need with a compoundcomprising a specifier (V) linked to two or more of the same ordifferent leaving groups (Z) via a self-eliminating multiple releasespacer or spacer system, which compound upon a single activation stepreleases at least two leaving groups, said activation step being theremoval or transformation of the specifier. Self-elimination spacers canalso be coupled to one another, so that more than one spacer isincorporated between specifier and leaving group. Hereinbelow, also theterm (self-elimination) spacer system is used, which comprises two ormore spacers, being either self-eliminating multiple release or singlerelease spacers, connected together. The present invention alsodescribes bifunctional linker systems comprising a (protected) specifiercontaining a reactive moiety that enables coupling to a targeting moiety(which then together with the initial specifier becomes a functionalisedspecifier V) on one side of the spacer (system). Further, thebifunctional linker systems comprise reactive moieties that enablecoupling to multiple leaving groups (for example parent drugs ordetectable molecules).

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows schematically the conversion of a spacer containing prodruginto the parent drug.

FIG. 2 shows schematically the liberation of multiple leaving groupmolecules from multiple release spacer containing compounds.

FIG. 3 shows schematically the liberation of 4 leaving group moleculesfrom a compound containing 2 generations of double release spacers.

FIG. 4 shows schematically the liberation of 9 leaving group moleculesfrom a compound containing 2 generations of triple release spacers.

FIG. 5 shows the principle of 1,6-elimination.

FIG. 6 shows the principle of 1,8-elimination.

FIG. 7 shows the proposed principle of elimination of 3 leaving groupsfrom a triple release spacer.

FIG. 8 shows the proposed principle of elimination of 2 leaving groupsfrom a double release spacer.

FIG. 9 shows the preparation of 2 model compounds containing a triplerelease spacer and 3 benzyl alcohol or phenethyl alcohol leaving groups.

FIG. 10 shows the preparation of a model compound containing a triplerelease spacer and 3 benzylamine leaving groups.

FIG. 11 shows the preparation of a model compound containing a triplerelease spacer and 3 propylamine leaving groups.

FIG. 12 shows the preparation of a prodrug containing a double releasespacer and 2 paclitaxel moieties.

FIG. 13 shows the preparation of a prodrug containing two generations ofdouble release spacers and 4 paclitaxel moieties.

FIG. 14 shows the preparation of a model compound containing a doublerelease spacer and 2 benzylamine leaving groups.

FIG. 15 shows the preparation of a doubly p-nitrophenyl carbonateactivated compound containing a double release spacer coupled to twosingle release spacers.

FIG. 16 shows the preparation of 3 model compounds containing a doublerelease spacer coupled to two single release spacers.

FIG. 17 shows the preparation of a model compound containing a singlerelease spacer coupled to benzylamine.

FIG. 18 shows the reference compounds dibenzyl carbonate and2′-O-cinnamyl oxycarbonylpaclitaxel.

FIG. 19 shows the reduction of the nitro group in the 2 model compoundscontaining 3 benzyl alcohol or 3 phenethyl alcohol leaving groups withthe concomitant liberation of all leaving groups.

FIG. 20 shows the reduction of the nitro group in the model compoundcontaining 3 benzylamine leaving groups.

FIG. 21 shows the removal of the Aloc group from the model compoundcontaining 3 propylamine leaving groups.

FIG. 22 shows the reduction of the nitro group in the prodrug containing2 paclitaxel moieties with the concomitant liberation of 2 molecules ofpaclitaxel.

FIG. 23 shows the reduction of the nitro group in the prodrug containing4 paclitaxel moieties with the concomitant liberation of 4 molecules ofpaclitaxel.

FIG. 24 shows the reduction of the nitro group in the model compoundcontaining 2 benzylamine leaving groups.

FIG. 25 shows the reduction of the nitro group in the 2 model compoundscontaining 2 p-methoxybenzylamine or 2 p-chlorobenzylamine leavinggroups.

FIG. 26 shows the reduction of the nitro group in the model compoundcontaining 2 phenethyl alcohol moieties with the concomitant liberationof 2 molecules of phenethyl alcohol.

FIG. 27 shows the reduction of the nitro group in the model compoundcontaining a benzylamine leaving group, with the concomitant liberationof benzylamine.

FIG. 28 shows the treatment of two reference compounds with zinc andacetic acid, which does not lead to any degradation of startingmaterial.

DETAILED DESCRIPTION OF THE INVENTION

In this invention a new technology is disclosed that can be used toinduce release of more than one leaving group following a singleactivation step. It can be applied for example to prepare prodrugs orconjugates, which can for example be used for targeting drugs todisease-related or organ-specific tissue or cells, for exampletumor-specific conjugates or prodrugs that are improved with respect toconjugates or prodrugs known thus far in that they release more than oneparent moiety after a single activation step. This aims at a therapeuticadvantage. The present invention is deemed to be applicable to all drugsthat need to be delivered at a specific target site where a specificdisease-related or specifically targeted biomolecule can convert theprodrug or conjugate into drugs or induce conversion of the prodrug orconjugate into drugs. This invention can furthermore find application in(non-specific) controlled release of compounds, with the aim ofenhancing characteristics of parent moiety release.

In another aspect, this invention can find application in a diagnosticassay process. An enzyme can be detected by a compound of this inventioncontaining a multiple release spacer or spacer system that isselectively activated by said enzyme to release multiple detectablemolecules (leaving groups), thus increasing the sensitivity of theassay.

In this invention, self-elimination multiple release spacers aredisclosed that enable release of multiple leaving group molecules upon asingle activation event. These self-elimination multiple release spacersare applicable in conjugates or prodrugs, for example anticancerprodrugs, and significantly enhance the amount of drug moleculesliberated per (enzymatic) activation, resulting in a potentialtherapeutic advantage.

In the generic structures throughout this description and in the claimsletters are used to define structural elements. Some of these letterscan be mistaken to represent an atom, such as C, N, O, P, B, F, S, V, W,I, Y. To avoid confusion whenever these letters do not represent an atomthey are given in bold typeface.

In the structures throughout this description and in the claimsmolecular structures or parts thereof are drawn. As usual in suchdrawings bonds between atoms are represented by lines, in some cases, toindicate stereochemistry, by bold or broken or wedged lines. Usually aline ending in space (a “loose” end), i.e. at one end not having anotherline or specific atom connected to it represents a CH₃ group. This iscorrect for the drawings representing the preferred compounds accordingto the invention hereinbelow. However for those structures representinga structural element of the compounds according to the invention, inparticular A, C, D, E, F, G, H, I, J. K, L, M, N, O, T, U and Y, a lineending in space indicates the position of attachment of anotherstructural element of the compound or conjugate. This includesattachment to V, S or Z. Also for the drawings representing thepreferred structural elements (W—)_(w)(X—)_(x)Cc,(W—)_(w)(X—)_(x)C(D_(d))_(c), (W—)_(w)(X—)_(x)C(D(E_(e))_(d))_(c) and(W—)_(w)(X—)_(x)C(D(E(F_(f))_(e))_(d))_(c) a line ending in spaceindicates the position of attachment of another structural element ofthe compound or conjugate, including attachment to V, S or Z. Analternative drawing of a line representing a bond to another structuralelement would be a drawing of a line with a wavy line perpendicular atthe “loose” end of the line.

Furthermore, the structures or parts thereof have been drawn, under theassumption that the structures are read from left to right, meaning thatthe specifier V is always located on the left side and the leavinggroups Z or the reactive moieties S are always located on the right sideof such structures.

According to the invention, two ‘branched self-elimination spacers’,herein also called ‘multiple release spacers’, have been developed thatare able to release multiple moieties. Spacers that are able to releaseonly a single moiety are called ‘unbranched spacers’ or ‘single releasespacers’. Spacers, either branched or unbranched, which self-eliminatethrough a 1,(4+2n)-elimination (n=0,1,2,3,4,5,6,7,8,9 or 10) (forexample 1,6-elimination, 1,8-elimination, or 1,10-elimination) arefurther called ‘electronic cascade’ spacers.

When a spacer able to release 2 leaving groups, hereinbelow called“double release spacer”, is used between specifier and leaving groups,two leaving group molecules are released per (enzymatic) activation(FIG. 2 a). When a spacer able to release 3 leaving groups, hereinbelowcalled “triple release spacer”, is used, three leaving group moleculeswill be released per activated compound or prodrug (FIG. 2 b).

When a spacer is connected to one or more other spacers via a directbond, this combination of spacers is referred to as ‘spacer system’.When at least one of these spacers is a multiple release spacer, it iscalled a ‘multiple release spacer system’.

An aniline-based spacer is a multiple release or single release spacerthat comprises an aromatic amino group on the lefthand side. A phenol-or thiophenol-based spacer is a multiple release or single releasespacer that comprises an aromatic hydroxy or thio group, respectively onthe lefihand side. Lefthand side refers to the position in the moleculeas shown in e.g. FIGS. 5, 6, 7 and 8.

Surprisingly it was found that it is required for more than one leavinggroup molecule to be released that the leaving groups are not aliphaticamine groups when only an aniline-based multiple release spacer isemployed. In other words the leaving groups (Z) should not be coupled tothe self-eliminating multiple release spacer via their aliphatic aminegroups if the spacer is an aniline-based multiple release spacer. In theexamples it is shown that if the leaving group Z is propylamine,p-methoxybenzylamine, benzylamine, or p-chlorobenzylamine, coupled viaits primary amine group, maximally one of these leaving groups isactually released (see examples 22, 23, 26, 27, and 28/FIGS. 20, 21, 24,and 25). When the spacer is a phenol-based or tiuophenol-based multiplerelease spacer or when the multiple release spacer is directly coupledto single release phenol- or thiophenol-based spacers or spacer systems,however, the leaving groups can also be coupled via their aliphaticamino group as all leaving groups are liberated upon activation.

Preferably the leaving groups Z are linked to the self-eliminatingmultiple release spacer via a group that possesses sufficientelectron-withdrawing capacity, such as for example O, S, aromatic N, oraliphatic N. It is important to note that in the context of thisinvention with aromatic N is meant a nitrogen atom covalently bound toan aromatic group such as for instance a (substituted) phenyl ring orother (hetero)aromatic group, but also aromatic N can mean a nitrogen inan aromatic ring such as for instance in a pyrrole ring or in animidazole ring. As disclosed in this invention, the leaving groups mustpossess better leaving group capabilities than aliphatic anines when themultiple release spacer is an aniline-based spacer. Propylamine,p-methoxybenzylamine, benzylamine, and even p-chlorobenzylamine did notpossess sufficient electron-withdrawing capacity to induce completerelease of all end groups when the spacer is an aniline-based spacer. Incontrast, benzyl alcohol, phenethyl alcohol, paclitaxel, and certainpara-substituted aniline derivatives all showed to be completelyreleased from double and triple release spacers disclosed in thisinvention. For example in case of the herein disclosed double releasespacers, when the para-substituted aniline derivative wasNH—C₆H4—CH₂—O—CO—NH—CH₂—R, multiple release did not take place. Incontrast, when the para-substituted aniline derivative wasNH—C₆H₄—CH═C(CH₂—O—CO—2′—O-paclitaxel)₂, multiple release did taleplace. When the multiple release spacer is a phenol- or thiophenol-basedmultiple release spacer or when the multiple release spacer is coupledto single release phenol- or thiophenol-based spacers, aliphatic aminesare also suitable leaving groups and release of all aliphatic amineleaving groups does occur. This can be reasoned based on the above asaliphatic alcohols prove to be suitable leaving groups (e.g., fromH₂N—C₆H₄—CH═C(CH₂—O—CO—2′—O-paclitaxel)₂ two molecules of paclitaxel arereleased) and therefore release of phenol-based spacers, to which analiphatic amine leaving group is connected, from a multiple releasespacer (e.g., H₂N—C₆H₄—CH═C(CH₂—O—C(O)O—C₆H₄—CH₂OC(O)NR¹R²)₂) shouldoccur by virtue of their increased leaving capability with respect toaliphatic alcohols. As elimination of aliphatic amines fromhydroxybenzyl-based spacer-aliphatic amine conjugates is known¹⁰,release of aliphatic amines from spacer systems in which the aliphaticamine is directly connected to a phenol-based spacer takes place aswell. The above shows that subtle changes can determine whether multiplerelease occurs or not. With all aliphatic amines, and even with certainpara-substituted aniline derivatives tested, release of more than oneleaving group did not take place when the spacer is an aniline-basedspacer, but occurs when the spacer is a phenol- or thiophenol-basedspacer, whereas with certain other para-substituted aniline derivatives,and with all hydroxyl leaving groups tested, complete elimination of allend groups did occur independent of the type of spacer used.

For at least two molecules to leave the compound according to theinvention the leaving group should be coupled via its oxygen, such asfor example its primary, secondary or tertiary alcohol, its phenol, orits phosphate, its sulphur, or its aromatic amine. In order for at leasttwo leaving groups that are coupled via their aliphatic amino group toleave from a conjugate comprising one multiple release spacer, thespacer should be a phenol- or thiophenol-based multiple release spaceror the spacer system comprising one aniline-based multiple releasespacer should also comprise single release phenol- or thiophenol-basedspacers directly connected to this multiple release spacer. The linkagebetween self-eliminating multiple release spacer (system) and leavinggroup can be described as carbonate, alkyl phosphate, oxycarbonylthio,carbaniate, or N-aryl-carbamate. In some cases, for example in case ofan aromatic alcohol leaving group coupled to a single releaseself-elimination spacer via an aryl ether linkage¹¹, the leaving groupcan also be linked to the spacer without an oxycarbonyl unit withoutcompromising self-elimination.

In one embodiment the compound comprises one self-eliminating multiplerelease spacer. In a further embodiment the compound comprises two ormore self-eliminating multiple release spacers. In yet a furtherembodiment the compound comprises three or more self-eliminatingmultiple release spacers.

The self-elimination multiple release spacers can also be coupled to oneanother (FIGS. 3 and 4). In this case compounds are obtained thatcontain multiple generations of multiple release spacers. If onemultiple release spacer is incorporated in the spacer system of thecompound according to the invention, the compound contains onegeneration of multiple release spacer. If two or more self-elimiatingmultiple release spacers are coupled to a first self-eliminatingmultiple release spacer, the resulting compound contains two generationsof multiple release spacers (second generation). If two or moreself-eliminating multiple release spacers are coupled to two or moremultiple release spacers that constitute the second generationself-eliminating multiple release spacers, the resulting compoundcontains three generations of multiple release spacers (thirdgeneration). If a compound according to the invention contains two ormore generations of self-eliminating multiple release spacers, thecompound possesses a dendritic (or dendrimeric) structure and can becalled a dendrimer. In one embodiment of the invention the compoundcomprises one self-eliminating multiple release spacer. In a furtherembodiment of the invention the compound comprises two or moregenerations of self-eliminating multiple release spacers. In yet afurther embodiment the compound comprises three or more generations ofself-eliminating multiple release spacers. For instance by coupling twoself-elimination double release spacers to a first self-eliminationdouble release spacer and optionally again coupling of fourself-elimination double release spacers to the second twoself-elimination double release spacers, a dendritic structure isconstructed. The number of leaving group molecules, for example drugmolecules, that can be bound per specifier is multiplied with everygeneration of self-elimination multiple release spacers incorporated.Each new generation of self-elimination multiple release spacers that iscoupled to the preceding generation multiplies the number of leavinggroup molecules that can eventually be present in the compound orconjugate by a factor that is equal to the number of leaving groups thatcan be bound to the multiple release spacer of the generation that isnewly incorporated. The final compound or prodrug (or bioconjugate) thatis obtained is a dendrimer.

Thus in a further embodiment of the invention the compound comprises aself-eliminating multiple release spacer system in the form of adendritic structure.

For at least two molecules to leave such a dendritic multiple releasespacer system, the leaving groups should be coupled via their oxygen,such as for example their primary, secondary or tertiary alcohol, theirphenol, or their phosphate, their sulphur, or their aromatic amine. Inorder for at least two leaving groups that are coupled via theiraliphatic amino group to leave, at least one generation of the dendriticmultiple release spacer system should comprise phenol- orthiophenol-based multiple release spacers or should be coupled to thenext generation of multiple release spacers or to the leaving groups viasingle release phenol- or thiophenol-based spacers. Preferably thehighest generation of multiple release spacers or spacer systems shouldbe phenol- or thiophenol-based.

In a preferred embodiment, when an aniline-, phenol- or thiophenol-basedmultiple release spacer (system) is used, the leaving groups are notaliphatic amines, but preferably for example O, S or aromatic N.

Dendrimers, also known as starburst polymers, are well-defined highlybranched tree-like macromolecules with a large number of end groups¹².One application of dendrimeric (or dendritic) structures that has beenexplored is drug delivery¹³. In the emerging field of dendrimeric drugdelivery, biologically active substances can be covalently linked todendrimeric end groups, or can be encapsulated inside dendrimers. In theexamples of the first case that have been reported thus far, each drugmolecule must be independently liberated via a chemical or biologicalcleavage step¹⁴. In the second case, specific physiological conditionsneed to change the folding and/or tertiary structure of the dendrimer,thereby releasing encapsulated material. Release of active substancesfrom dendrimers can be induced by pH¹⁵, incorporation of photosensitiveunits¹⁶, or by enzymatic cleavage¹⁷.

The novel self-elimination multiple release spacers disclosed hereinenable complete release of multiple leaving groups, for examplebiologically active substances, that are covalently linked to thedendrimeric end groups in such a way that a single activation issufficient to release all end groups (FIGS. 3 and 4). A single chemicalor biological activating event should lead to a cascade ofself-elimination reactions thereby releasing multiple leaving groups.Dendrimers with these properties may be useful for several applications,and are considered particularly useful in the area of diagnostics andthe area of drug delivery, for example in anticancer therapy, wheretumor-selective degradation of the dendrimeric material is desired.Multiple drug molecules can be site-specifically released by employmentof only one targeting or drug delivery device. Compounds that containtwo or more generations of multiple release self-elimination spacers,optionally connected to each other via single release spacers or spacersystems are further called ‘cascade dendrimers’.

Several applications are imaginable for the multiple release spacers orspacer systems or cascade dendrimers of this invention. Firstly,bioactive compounds can be site-specifically delivered. The concept canbe applied to anticancer agents, but also antibiotics can beincorporated as end groups in a cascade dendrimer that is activated forexample by bacterial enzymes, such as for example β-lactamase. Inaddition, cascade dendrimers may be applicable in the agrochemical fieldfor release of pesticides¹⁸. Through sophisticated synthesis, cascadedendrimers may be prepared that contain two or more different parentcompounds. This may be interesting when it is considered thatcombination therapy emerges as a clinically important mode of treatmentfor diseases such as cancer, bacterial diseases, and HIV.

Furthermore, biodegradable dendrimers might be interesting for theproduction of biodegradable materials such as plastics, or may be usedas devices that enable the controlled or slow release of drugs byincorporation of, for example, an enzymatically degradable sequence. Inaddition, cascade dendrimers may be used for diagnostic purposes¹⁹. Theycould serve as an amplification mechanism in diagnostic assays, seehereinbelow.

Several strategies have evolved to improve the specificity of anticancerdrugs, the prodrug concept being one of them^(20,21). A prodrug is aninactive derivative of an active drug, which is site-specificallyactivated to release the active parent drug. Prodrugs that are activatedby tumor-specific or tumor-targeted enzymes have shown promisingresults²². Alternatively, prodrugs of antitumor agents coupled topolymers, as already reported by Ringsdorf²³, provide another targetingprinciple. Polymers, including dendrimers, can passively lead totumnor-specificity, due to the enhanced permeability and retentioneffect (EPR)²⁴. This effect retains polymeric material with anapproximate molecular weight >40 kD inside tumor tissue due todiscontinuous (leaky) and poorly formed tumor endothelium and poorlymphatic drainage. Frequently used polymeric carriers for drug deliveryare poly[N-(2-hydroxypropyl)methacrylamide] (poly-HPMA)²⁵, poly-glutamicacid, for example poly-L-glutamic acid (PG), and poly(ethylene) glycol(PEG)²⁶. Application of dendrimers in drug delivery can be preferred toother polymers because, by definition, dendrimers are not aspolydisperse as conventional polymers. In general, they can be obtainedas homogeneous compounds more easily than polymers, a fact that mayfacilitate approval of dendrimeric material for medicinal purposes.

An important advantage of cascade dendrimers over conventionaldendrimers or polymers would be that they would be selectivelydegradable, thus clearable from the body. Clearance of polymers from thebody is a limiting factor in current polymeric drug delivery systems, asthe maximum molecular weight of synthetic macromolecules that can becleared from the body ranges from 25 to 45 kD²⁷. Cascade dendrimers asdisclosed herein can be considered as an alternative for self-assemblingdendrimeric systems²⁸ that also possess these desirable properties ofdegradability.

Thus, the multiple release dendrimeric conjugates and prodrugs of thisinvention containing covalently bound anticancer drug molecules as endgroups and activated by a specific enzyme that is localized in the tumorenvironment or by any other specific chemical or biological event wouldcombine a number of desirable properties for a tumor-selectiveanticancer compound.

More specifically the invention relates to compounds of the formulasV—(W—)_(w)(X—)_(x)C((A—)_(a)Z)_(c),V—(W—)_(w)(X—)_(x)C(D((A—)_(a)Z)_(d))_(c),V—(W—)_(w)(X—)_(x)C(D(E((A—)_(a)Z)_(e))_(d))_(c), andV—(W—)_(w)(X—)_(x)C(D(E(F((A—)_(a)Z)_(f))_(e))_(d))_(c),wherein:

-   -   V is selected from [O] and a specifier which is removed or        transformed by a chemical, photochemical, physical, biological,        or enzymatic activation, optionally after prior binding to a        receptor;        (W—)_(w)(X—)_(x)C((A—)_(a))_(c),        (W—)_(w)(X—)_(x)C(D((A—)_(a))_(d))_(c),        (W—)_(w)(X—)_(x)C(D(E((A—)_(a))_(e))_(d))_(c), and        (W—)_(w)(X—)_(x)C(D(E(F((A—)_(a))_(f))_(e))_(d))_(c)        are self-eliminating multiple release spacers or spacer systems;    -   W and X are each a single release 1,(4+2n) electronic cascade        spacer, being the same or different;    -   A is a cyclization elimination spacer;    -   C, D, E, and F are each a self-eliminating multiple release        spacer or spacer system that upon activation can maximally        release c, d, e, and f leaving groups, respectively;    -   each Z is independently a leaving group or H or OH or a reactive        moiety;    -   a is 0 or 1;    -   c, d, e, and f are independently an integer from 2 (included) to        24 (included);    -   w and x are independently an integer from 0 (included) to 5        (included);    -   n is an integer of 0 (included) to 10 (included).

In a further embodiment, the invention relates to compounds of theformulasV—(W—)_(w)(X—)_(x)C((A—)_(a)S)_(c),V—(W—)_(w)(X—)_(x)C(D((A—)_(a)S)_(d))_(c),V—(W—)_(w)(X—)_(x)C(D(E((A—)_(a)S)_(e))_(d))_(c), andV—(W—)_(w)(X—)_(x)C(D(E(F((A—)_(a)S)_(f))_(e))_(d))_(c),wherein:

-   -   V, W, X, C, D, E, F, A, w, x, c, d, e, f, and a are as defined        above and each S independently has no meaning or is H, OH, or a        reactive moiety that allows for coupling the multiple release        spacer system to leaving groups Z, which may be the same or        different, to afford compounds        V—(W—)_(w)(X—)_(x)C((A—)_(a)Z)_(c),        V—(W—)w(X)_(x)C(D((A—)_(a)Z)_(d))_(c),        V—(W—)_(w)(X—)_(x)C(D(E((A—)_(a)Z)_(e))_(d))_(c), and        V—(W—)_(w)(X—)_(x)C(D(E(F((A—)_(a)Z)_(f))_(e))_(d))_(c),        respectively.

A compound or prodrug (or (bio)conjugate) according to this inventioncomprises a specifier V, which is meant to consist of a group that canbe site specifically removed or transformed and that is covalentlyattached to at least two therapeutic or diagnostic moieties Z or to atleast two reactive moieties S via the novel self-eliminating multiplerelease spacer or spacer system (W—)_(w)(X)_(x)C((A—)_(a))_(c) or(W—)_(w)(X—)_(x)C(D((A—)_(a))_(d))_(c) or(W—)_(w)(X—)_(x)C(D(E((A—)_(a))_(e))_(d))_(c) or(W—)_(w)(X—)_(x)C(D(E(F((A—)_(a))_(f))_(e))_(d))_(c). Theseself-eliminating multiple release spacers or spacer systems possessmultiple sites for attachment of moieties Z or moieties S.

According to a preferred embodiment of the invention, theself-elimination multiple release spacers or spacer systems C, D, E, andF are independently selected from compounds having the formula

wherein

-   -   B is selected from NR¹, O, and S;    -   P is C(R²)(R³)Q—(W—)_(w)(X—)_(x);    -   Q has no meaning or is —O—CO—;    -   W and X are each a single release 1,(4+2n) electronic cascade        spacer, being the same or different;    -   G, H, I, J, K, L, M, N, and O are independently selected from        compounds having the formula:        wherein R¹, R², R³, R⁴, and R⁵ independently represent H, C₁₋₆        alkyl, C₃₋₂₀ heterocyclyl, C₅₋₂₀ aryl, C₁₋₆ alkoxy, hydroxy        (OH), amino (NH₂), mono-substituted amino (NR_(x)H),        di-substituted amino (NR_(x) ¹R_(x) ²) nitro (NO₂), halogen,        CF₃, CN, CONH₂, SO₂Me, CONHMe, cyclic C₁₋₅ alkylamino,        imidazolyl, C₁₋₆ alkylpiperazinyl, morpholino, thiol (SH),        thioether (SR_(x)), tetrazole, carboxy (COOH), carboxylate        (COOR_(x)), sulphoxy (S(═O)₂OH), sulphonate (S(═O)₂OR_(x)),        sulphonyl (S(═O)₂R_(x)), sulphixy (S(═O)OH), sulphinate        (S(═O)OR_(x)), sulphinyl (S(═O)R_(x)), phosphonooxy        (OP(═O)(OH)₂), and phosphate (OP(═O)(OR_(x))₂), where R_(x),        R_(x) ¹ and R_(x) ² are independently selected from a C₁₋₆ alkyl        group, a C₃₋₂₀ heterocyclyl group or a C₅₋₂₀ aryl group, two or        more of the substituents R¹, R², R³, R⁴, and R⁵ optionally being        connected to one another to form one or more aliphatic or        aromatic cyclic structures, or    -   G, J, and M may also be selected from the group of P and        hydrogen with the proviso that if two of G, J, and M are        hydrogen, the remaining group must be    -   or be    -   and at the same time be conjugated to    -   g, h, i, j, k, l, m, n, o, h′, g′, k′, j′, n′, m′ are        independently 0, 1, or 2 with the provisos that    -   if G=hydrogen or P, g, h, i, h′, and g′ all equal 0;    -   if J=hydrogen or P, j, k, l, k′, and j′ all equal 0;    -   if M=hydrogen or P, m, n, o, n′, and m′ all equal 0;    -   if G, H, I, J, K, L, M, N, or O is    -   then g+g′=1, h+h′=1,i=1, j+j′=1, k+k′=1, l=1,m+m′=1,n+n′=1, or        o=1, respectively;    -   if G, H, I, J, K, L, M, N, or O is    -   then g+g′=2, h+h′=2, i=2,j+j′=2, k+k′=2, l=2, m+m′=2, n+n′=2, or        o=1, respectively;    -   if g′=0 and G is not hydrogen or P, then h, h′, and i equal 0        and g>0;    -   if g=0 and G is not hydrogen or P, then g′>0;    -   if g′>0 and h′=0, then i=0 and h>0;    -   if g′>0 and h=0, then h′>0 and i>0;    -   if j′=0 and J is not hydrogen or P, then k, k′, and 1 equal 0        and j>0;    -   if j=0 and J is not hydrogen or P, then j′>0;    -   if j′>0 and k′=0, then l=0 and k>0;    -   if j′>0 and k=0,then k′>0 and l>0;    -   if m′=0 and M is not hydrogen or P, then n, n′, and o equal 0        and m>0;    -   if m=0 and M is not hydrogen or P, then m′>0;    -   if m′>0 and n′=0, then o=0 and n>0;    -   if m′>0 and n=0,then n′>0 and o>0;    -   w and x are independently an integer from 0 (included) to 5        (included);    -   with the proviso that    -   if the compound contains only C and no D, no E, and no F are        present, and B=NR¹, and G and M are H, and g, h, i, h′, g′, k,        l, k′, l′, m, n, o, n′, and m′ are 0, and J=        and j=2, and Q=—O—CO—, and w and x are 0, and R¹, R², R³, and R⁴        are H, then at least one of the leaving groups Z is not        connected to Q via an aliphatic amino group.

According to another preferred embodiment of the invention the 1,(4+2n)electronic cascade spacers W and X are independently selected fromcompounds having the formula

Q′=—R⁵C═CR⁶—, S, O, NR⁵, —R⁵C═N—, or —N═CR⁵—B=NR⁷, O, SP=C(R³)(R⁴)Qwherein

-   -   Q has no meaning or is —O—CO—;    -   t, u, and y are independently an integer of 0 to 5;    -   T, U, and Y are independently selected from compounds having the        formula:        wherein R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, and R⁹ independently        represent H, C₁₋₆ alkyl, C₃₋₂₀ heterocyclyl, C₅₋₂₀ aryl, C₁₋₆        alkoxy, hydroxy (OH), amino (NH₂), mono-substituted amino        (NR_(x)H) di-substituted amino (NR_(x) ¹R_(x) ²), nitro (NO₂),        halogen, CF₃, CN, CONH₂, SO₂Me, CONHMe, cyclic C₁₋₅ alkylamino,        imidazolyl, C₁₋₆ alkylpiperazinyl, morpholino, thiol (SH),        thioether (SR_(x)), tetrazole, carboxy (COOH), carboxylate        (COOR_(x)), sulphoxy (S(═O)₂OH), sulphonate (S(═O)₂OR_(x)),        sulphonyl (S(═O)₂R_(x)), sulphixy (S(═O)OH), sulphinate        (S(═O)OR_(x)), sulphinyl (S(═O)R_(x)), phosphonooxy        (OP(═O)(OH)₂), and phosphate (OP(═O)(OR_(x))₂), where R_(x),        R_(x) ¹ and R_(x) ² are independently selected from a C₁₋₆ alkyl        group, a C₃₋₂₀ heterocyclyl group or a C₅₋₂₀ aryl group, two or        more of the substituents R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, or R⁹        optionally being connected to one another to form one or more        aliphatic or aromatic cyclic structures.

In a preferred embodiment leaving groups Z are linked to theself-eliminating multiple release spacer or spacer system via an O, S oraromatic N of the leaving group.

In order to farther clarify the above mentioned formulae, compound 26depicted in FIG. 13 will serve as an example. This compound is acompound of the formula V—(W—)_(w)(X—)_(x)C(D((A—)_(a)Z)_(d))_(c). Itcontains two generations of double release spacers, to which 4 endgroups Z have been coupled. The specifier V is [O], which means that V—Bis an oxidized form of B (part of C). Reduction of this functionalityinduces self-elimination. Such moiety V—B can be chemically reduced, butit can also be reduced under physiological conditions under hypoxicconditions, or by a nitroreductase. Z is paclitaxel. No single releasespacer is incorporated in between the specifier [O] and the multiplerelease spacers, which means that w=x=0. C is the double release spacercontaining the benzylidene-propane-1,3-bis-oxycarbonyl unit, whicheliminates according to the principle depicted in FIG. 8. No cyclizationelimination spacer is present, which means that a=0. Because two doublerelease spacers have been coupled to the first double release spacer,d=c=2.

In general, multiple release spacers disclosed in this invention possessmultiple release characteristics because they are branched on twolevels; i) branching on the aromatic ring where both ortho andpara-substituents with respect to the location of the specifier can beintroduced, and ii) branching by incorporation of additional conjugateddouble bonds at para and/or ortho positions of the aromatic rings, wherethe terminal C-atom of the double bond provides a branching point wheretwo leaving groups can be attached. Theoretically, these two elementsfor branching self-eliminating spacers to obtain multiple releasespacers or spacer systems can be infinitely extended.

In addition to incorporation of one or more (generations of)self-eliminating multiple release spacers, it may also be desirable toincorporate a single release, also called unbranched, self-eliminatingspacer or spacer system in between the specifier and the first multiplerelease spacer. This may facilitate the activation step. This singlerelease self-eliminating spacer or spacer system is described by—(W—)_(w)(X—)_(x). Furthermore, it may be desirable to add singlerelease self-eliminating spacers or spacer systems to a previousgeneration, coupled to a next generation of multiple release spacers(thus obtaining a previous generation of multiple release spacersystems), or to the highest generation of multiple release spacer(s)before leaving groups Z in order to increase the surface area of thefinal conjugate. This may increase the number of end groups that can beaccommodated and it may also increase the fmal size of the conjugate,which may go together with certain advantages, such as for examplebenefiting from the Enhanced Permeability and Retention (EPR) effect.Furthermore, when only aniline-based multiple release spacers are used,connection of single release phenol- or thiophenol-based spacers to ageneration of multiple release spacers or, preferably, to the highestgeneration of multiple release spacers before leaving groups Z enablesthe release of two or more leaving groups Z that are coupled with theiraliphatic amino group to the multiple release spacer system. When onlyaniline-based multiple release spacers are used and no single releasephenol- or thiophenol-based spacers are incorporated into the multiplerelease spacer system to which leaving groups Z are coupled with theiraliphatic amino group only 1 Z group will be released. This is also truefor incorporation of single release aniline-based spacer(s) in themultiple release spacer system to which leaving groups Z are coupledwith their aliphatic amino group. Single release spacers that are addedto a generation of multiple release spacers, or to the highestgeneration of multiple release spacer(s) before leaving groups Z orreactive moieties S are represented in the above formulae by—(W—)_(w)(X—)_(x) in P, which is a part of C, D, E, and F.

In general, aniline-based single release or multiple release spacers arepreferred over phenol- or thiophenol-based single release or multiplerelease spacers. The above shows, however that it might sometimes bebeneficial to include phenol- or thiophenol-based single release spacerin the multiple release spacer system. For the same reason, it issometimes beneficial to include phenol- or thiophenol-based multiplerelease spacers. In general, to release two or more Z groups that areconnected to the multiple release spacer or spacer system via analiphatic amino group, at least one, preferably all, multiple releasespacers or spacer systems of either generation C, D (if present), E (ifpresent), or F (if present), preferably the one(s) connected to eitherA, Z, or S, have to be phenol- or thiophenol-based multiple releasespacers or spacer systems, meaning that

-   -   i) B=O or S for at least one, preferably all, multiple release        spacers in that generation, or    -   ii) when B=N for all multiple release spacers in said        generation, at least one single release spacer is connected to        at least two branches of at least one, preferably all, multiple        release spacer in said generation, and B=O or S for at least two        of those single release spacers.

In the formulae above, Q is preferably O—CO, but it may also have nomeaning. For example, a compound with an aryl ether linkage betweenself-elimination spacer and leaving group, where the oxycarbonylfunction is lacking (Q has no meaning), has been reported to undergoself-eliniation as well¹¹.

The principle of 1,6-elimination, as such developed by Carl et al. in1981, can be considered one of the most versatile self-elininationprinciples that can be used in prodrug design. According to thisprinciple, spacer elimination proceeds via the mechanism depicted inFIG. 5. This particular elimination process has proven to be verysuccessful when applied in the prodrug concept. Spacers thatself-eliminate through an electronic cascade sequence as indicated inFIGS. 5 and 6 generally show much faster half-lives of elimination thando spacers that eliminate via a cyclization reaction. This is asignificant difference between cyclization spacers and electroniccascade spacers.

Elongated self-elimination spacers can be incorporated between acleavable moiety and leaving group, for example between specifier anddrug as disclosed in WO 02/083180 (see also ref. 29). Increasing thelength of self-elimination spacers may have (an) additionaladvantage(s). Self-elimination spacers with increased length mayincrease the rate and/or efficiency of activation of conjugate orprodrug. As a result, drug release characteristics may be enhancedthrough long spacers. The self-elimination spacers or spacer systems ofthe present invention can also be longer than conventionalself-elimination spacers, such as for example the 1,6-eliminationspacer, because they are elongated themselves or because multipleself-elimination linkers are combined within one multiple release spacersystem. Thus, the spacers or spacer systems of the present invention canbe branched, but in addition they can be elongated. Both factors (lengthand degree of branching) of a spacer or spacer system must be carefullyconsidered when designing conjugates or prodrugs. A high degree ofbranching may be disadvantageous for the efficiency of activation of theconjugate or prodrug, especially when the branching points are in closeproximity to the site of activation. When a high degree of branching isdesirable in order to accommodate many leaving groups Z, it may bebeneficial to incorporate single release self-elimination spacers inbetween the specifier V and the branching point(s). It may increase theefficiency of activation.

Connection of single release spacer(s) to generations of multiplerelease spacers may enable an increase of the total number of end groupsthat can be incorporated because the surface of the outer sphere isincreased by incorporation of additional single release spacer(s), andas a result more end groups (for example drugs) can be accommodated. Toobtain a conjugate or prodrug that possesses the desired properties, aconsidered choice of a specific multiple release spacer or spacer systemoptionally including one or more single release self-eliminationspacers, must be made.

The novel self-eliminating multiple release spacers disclosed in thisinvention may be coupled to one another to yield multiple release spacersystems in which the spacers are coupled through aryl-carbamatefunctions, by employing hydroxybenzotriazole as a catalyst to couple anaromatic amine to a p-nitrophenyl carbonate.

The invention is in another aspect related to compounds of theabove-mentioned formulas wherein A is a cyclization spacer, furthercalled ω-amino aminocarbonyl cyclization spacer having a formulaselected from

wherein:

-   -   a is an integer of 0 or 1; and    -   b is an integer of 0 or 1; and    -   c is an integer of 0 or 1; provided that    -   a+b+c=2 or3;    -   and wherein R¹ and R² independently represent H, C₁₋₆ alkyl,        said alkyl being optionally substituted with one or more of the        following groups: hydroxy (OH), ether (OR_(x)), amino (NH₂),        mono-substituted amino (NR_(x)H), di-substituted amino (NR_(x)        ¹R_(x) ²), nitro (NO₂), halogen, CF₃, CN, CONH₂, SO₂Me, CONHMe,        cyclic C₁₋₅ alkylamino, imidazolyl, C₁₋₆ alkylpiperazinyl,        morpholino, thiol (SH), thioether (SR_(x)), tetrazole, carboxy        (COOH), carboxylate (COORs), sulphoxy (S(═O)₂OH), sulphonate        (S(═O)₂OR_(x)), sulphonyl (S(═O)₂R_(x)), sulphixy (S(═O)OH),        sulphinate (S(═O)OR_(x)), sulphinyl (S(═O)R_(x)), phosphonooxy        (OP(═O)(OH)₂), and phosphate (OP(═O)(OR_(x))₂), where R_(x),        R_(x) ¹ and R_(x) ² are selected from a C₁₋₆ alkyl group, a        C₃₋₂₀ heterocyclyl group or a C₅₋₂₀ aryl group;    -   R² has no meaning when S has no meaning, which means that a        double bond is present between the rightmost nitrogen atom and        the carbonyl group (N═C═O); and R³, R⁴, R⁵, R⁶, R⁷, and R⁸        independently represent H, C₁₋₆ alkyl, C₃₋₂₀ heterocyclyl, C₅₋₂₀        aryl, C₁₋₆ allkoxy, hydroxy (OH), amino (NH₂), mono-substituted        amino (NR_(x)H), di-substituted amino (NR_(x) ¹R_(x) ²), nitro        (NO₂), halogen, CF₃, CN, CONH₂, SO₂Me, CONHMe, cyclic C₁₋₅        alkylamino, imidazolyl, C₁₋₆ alkylpiperazinyl, morpholino, thiol        (SH), thioether (SR_(x)), tetrazole, carboxy (COOH), carboxylate        (COOR_(x)), sulphoxy (S(═O)₂OH), sulphonate (S(═O)₂OR_(x)),        sulphonyl (S(═O )₂R_(x)), sulphixy (S(═O)OH), sulphinate        (S(═O)OR_(x)), sulphinyl (S(═O)R_(x)), phosphonooxy        (OP(═O)(OH)₂), and phosphate (OP(═O)(OR_(x))₂), where R_(x),        R_(x) ² and R 2 are selected from a C₁₋₆ alkyl group, a C₃₋₂₀        heterocyclyl group or a C₅₋₂₀ aryl group; and    -   wherein R¹, R², R³, R⁴, R⁵, R⁶, R⁷, and R⁸ can be a part of one        or more aliphatic or aromatic cyclic structures, two or more of        the substituents R¹, R², R³, R⁴, R⁵, R⁶, R⁷, or R⁸ optionally        being connected to one another to form one or more aliphatic or        aromatic cyclic structures.

The ω-amino group of the ω-amino aninocarbonyl cyclization spacer A doesnot need to be in close proximity to a particular electron-withdrawingsubstituent nor does it need to be an aromatic nitrogen, when it isconnected to a multiple release spacer system that contains phenol- orthiophenol-based spacers in one generation, preferably in the highestgeneration (which is the one connected to A, Z, or S).

The ω-amino group of the ω-amino aminocarbonyl cyclization spacer shouldpossess sufficient leaving group ability. In a preferred embodiment theω-amino group of the ω-amino aminocarbonyl cyclization spacer is anN-aryl or an aliphatic N wherein the N is positioned in proximity of anelectron-withdrawing group, such that the electron-withdrawing group canproperly exert its electron-withdrawing property so that the leavinggroup ability of the ω-amino group is increased. The nucleophilicity ofthe leaving amino group should however be sufficient to allow for asufficiently fast cyclization reaction.

In a further embodiment the invention relates to a compound, whereingroup A is an ω-amino aminocarbonyl cyclization spacer, and Z is amoiety coupled via its hydroxyl group to A.

Preferred compounds according to the invention are those wherein(W—)_(w)(X—)_(x)C_(c),(W—)_(w)(X—)_(x)C(D_(d))_(c),(W—)_(w)(X—)_(x)C(D(E_(e))_(d))_(c) or(W—)_(w)(X—)_(x)C(D(E(F_(f))_(e))_(d))_(c)is selected from the group consisting of

and from the compounds depicted above wherein single release1,6-elimination p-aminobenzyloxycarbonyl spacer(s) are replaced bysingle release 1,8-elimination p-aminocinnamyloxycarbonyl spacer(s).

In a further embodiment the compounds depicted in the previous paragraphfurther comprise ω-amino aminocarbonyl cyclization spacers A.

The invention also relates to the self-eliminating multiple releasespacers or spacer systems per se as described in this invention. Theinvention thus relates to compounds as defined above without specifier Vand with Z or S is H or OH, exemplified by for instance aminotriol 16(FIG. 11) and aminodiol 23 (FIG. 13), extending to spacer systemscomprising more than one spacer.

Also the invention relates to derivatives of the self-eliminatingmultiple release spacers or spacer systems per se that can be used forfacilitating coupling with specifiers, leaving groups, reactive moietiesor spacers. For instance an amino group may be derivatized as itsisocyanate and an alcohol group may be derivatized as its p-nitrophenylcarbonate. Other derivatizations are well within the knowledge of theskilled person. One end of the spacer or spacer system must be able toreact with the specifier, for example the tripeptide that is a substratefor plasmin. Typically, this end of the spacer or spacer system is anamino group or a hydroxyl group, but it can also be anotherfunctionality. The functionalities at the other end of the linker orlinker system must be able to react with the leaving group Z (the drug).Typically, these ends of the multiple release spacer or spacer systemare hydroxyl groups, but they can also be other functionalities. In oneembodiment, these functionalities react with the hydroxyl group of thedrug to form for example carbonate linkages between linker and drug. Inanother embodiment, these functionalities react with the aromatic aminogroup of the drug to form for example N-aryl carbamate linkages betweenlinker and drug. In again another embodiment, these functionalitiesreact with the sulfhydryl group of the drug to form for exampleoxycarbonylthio linkages between linker and drug. In again anotherembodiment these functionalities react with the carboxylic acid group ofthe drug. If spacers A are included in the spacer system, typically thehydroxyl group of the drug is reacted with A to form carbamate linkagesbetween A and Z.

In the compounds of the invention the specifier V typically contains asubstrate molecule that is specifically cleaved by an enzyme present inthe vicinity of or inside the target cells, for example tumor cells.More preferably, the specifier V contains a substrate that isspecifically cleaved by an enzyme present at elevated levels in thevicinity of or inside the target cells as compared to other parts of thebody, and most preferably the enzyme is present only in the vicinity ofor inside the target cells. In an embodiment suitably the specifier Vconstitutes a targeting moiety in order to target the compounds tospecific cells.

In the compounds of the invention, the specifier V may also contain areactive moiety that can react with a targeting moiety that can targetthe resulting compounds to the target site by selective binding to areceptor or other receptive moiety associated with a given target cellpopulation or by causing accumulation of the compounds of the inventionin the vicinity of or inside the target cells by another mechanism. Incase these compounds contain two or more reactive moieties S, thecompounds are from hereon called “bifunctional linkers”. When thecompounds comprise two or more moieties Z, the compounds are from hereoncalled “monofunctional spacer-leaving group conjugates”. Bothbifunctional linkers and monofunctional spacer-leaving group conjugatescan be used to prepare prodrugs of the present invention that contain aspecifier with a targeting group as well as a substrate specificallycleavable at the target site, a multiple release spacer system and twoor more leaving groups Z. Although in this invention spacers and spacersystems are described that are able to release multiple leaving groups,it is obvious that monofunctional spacer-leaving group conjugates andbifunctional linkers can also be designed that contain single releasespacers and spacer systems, such as for example described in WO 02/83180and EP 1243276, which are incorporated by reference.

In an aspect of the invention, the reactive moiety in V is reacted witha nucleophilic group on a targeting moiety, e.g., a thiol group, anamino group, or an aldehyde group, to form a new specifier V thatcontains a targeting moiety.

In a preferred aspect of the invention, the reactive moiety in V isreacted with a nucleophilic group on a protein or protein fragment,e.g., a thiol group, an amino group, or an aldehyde group, to form a newspecifier V that contains a protein or a protein fragment as thetargeting moiety. In a more preferred aspect of the invention, thereactive moiety in V is reacted with a nucleophilic group on an antibodyor antibody fragment, e.g., a thiol group, an amino group, or analdehyde group, to form a new specifier V that contains an antibody orantibody fragment as the targeting moiety. In another more preferredaspect of the invention, the reactive moiety in V is reacted with anucleophilic group on a peptide vector or receptor-binding moiety, e.g.,a thiol group, an amino group, or an aldehyde group, to form a newspecifier V that contains a peptide vector or receptor-binding moiety asthe targeting moiety.

In a preferred embodiment the reactive moiety in V is, withoutlimitation,

wherein X is een leaving group. These reactive moieties can be used tocouple a targeting moiety having a nucleophilic group, e.g., a thiolgroup, to the specifier V having such a reactive moiety to form a newspecifier V that contains a targeting moiety. In a more preferredembodiment of the invention, the reactive moiety in V is, withoutlimitation,

and is reacted with a nucleophilic group on a protein or proteinfragment, e.g., a thiol group, to form a new specifier V that contains aprotein or a protein fragment as the targeting moiety. In another morepreferred embodiment of the invention, the reactive moiety in V is,without limitation,

and is reacted with a nucleophilic group on an antibody or antibodyfragment, e.g., a thiol group, to form a new specifier V that containsan antibody or antibody fragment as the targeting moiety. In anothermore preferred embodiment of the invention, the reactive moiety in V is,without limitation,

and is reacted with a nucleophilic group on a peptide vector orreceptor-binding moiety, e.g., a thiol group, to form a new specifier Vthat contains a peptide vector or a receptor-binding moiety as thetargeting moiety.

In another preferred embodiment, the reactive moiety in V is, withoutlimitation, an activated ester such as a N-hydroxysuccinimide ester, ap-nitrophenyl ester, a pentafluorophenyl ester, and further anisothiocyanate, isocyanate, anhydride, acid chloride, sulfonyl chloride,or aldehyde. These reactive moieties can be used to couple a targetingmoiety having a nucleophilic group, e.g., an amino group, to thespecifier V having such a reactive moiety to form a new specifier V thatcontains a targeting moiety. In a more preferred embodiment of theinvention, the reactive moiety in V is, without limitation, an activatedester such as a N-hydroxysuccinimide ester, a p-nitrophenyl ester, apentafluorophenyl ester, and further an isothiocyanate, isocyanate,anhydride, acid chloride, sulfonyl chloride, or aldehyde, and is reactedwith a nucleophilic group on a protein or protein fragment, e.g., anamino group, to form a new specifier V that contains a protein or aprotein fragment as the targeting moiety. In another more preferredembodiment of the invention, the reactive moiety in V is, withoutlimitation, an activated ester such as a N-hydroxysuccinimide ester, ap-nitrophenyl ester, a pentafluorophenyl ester, and further anisothiocyanate, isocyanate, anhydride, acid chloride, sulfonyl chloride,or aldehyde, and is reacted with a nucleophilic group on an antibody orantibody fragment, e.g., an amino group, to form a new specifier V thatcontains an antibody or antibody fragment as the targeting moiety. Inanother more preferred embodiment of the invention, the reactive moietyin V is, without limitation, an activated ester such as aN-hydroxysuccinimide ester, a p-nitrophenyl ester, a pentafluorophenylester, and further an isothiocyanate, isocyanate, anhydride, acidchloride, sulfonyl chloride, or aldehyde, and is reacted with anucleophilic group on a peptide vector or receptor-binding moiety, e.g.,an amino group, to form a new specifier V that contains a peptide vectoror a receptor-binding moiety as the targeting moiety.

In another preferred embodiment, the reactive moiety in V is, withoutlimitation, an amino group or a hydrazine group. These reactive moietiescan be used to couple a targeting moiety having a nucleophilic group,e.g., an aldehyde group, to the specifier V having such a reactivemoiety to form a new specifier V that contains a targeting moiety. In amore preferred embodiment of the invention, the reactive moiety in V is,without limitation, an amino group or a hydrazine group, and is reactedwith a nucleophilic group on a protein or protein fragment, e.g., analdehyde group, to form a new specifier V that contains a protein or aprotein fragment as the targeting moiety. In another more preferredembodiment of the invention, the reactive moiety in V is, withoutlimitation, an amino group or a hydrazine group, and is reacted with anucleophilic group on an antibody or antibody fragment, e.g., analdehyde group, to form a new specifier V that contains an antibody orantibody fragment as the targeting moiety. In another more preferredembodiment of the invention, the reactive moiety in V is, withoutlimitation, an amino group or a hydrazine group, and is reacted with anucleophilic group on a peptide vector or receptor-binding moiety, e.g.,an aldehyde group, to form a new specifier V that contains a peptidevector or a receptor-binding moiety as the targeting moiety.

The specifier V may also contain a moiety that targets the compounds ofthe invention to the target site by selective binding to a receptor orother receptive moiety associated with a given target cell population orby causing accumulation of the compounds of the invention in thevicinity of or inside the target cells by another mechanism. Thistargeting moiety may, for example, be bombesin, transferrin, gastrin,gastrin-releasing peptide, a molecule that specifically binds α_(v)β3and/or α_(v)β₅-integrin receptors, such as RGD-containing peptides,platelet-derived growth factor, IL-2, IL-6, a tumor growth factor,vaccinia growth factor, insulin and insulin-like growth factors I en II,an antigen-recognizing immunoglobulin or an antigen-recognizing fragmentthereof, or a carbohydrate. Preferably, that antigen recognized by theimmunoglobulin (or fragment thereof) is specific for the target cells,e.g. a tumor-specific antigen.

In one embodiment, the specifier V contains a di-, tri-, or oligopeptidewhich consists of an amino acid sequence specifically recognized andthus cleavable by a protease, for example plasmin, a cathepsin,cathepsin B, prostate-specific antigen (PSA), urokinase-type plasminogenactivator (u-PA), or a member of the family of matrixmetalloproteinases, present in the vicinity of or inside the targetcells, for example tumor cells. Typically, the specifier V contains asubstrate for the serine protease plasmin.

In another embodiment, V contains a substrate for one or more of thecathepsins, typically cathepsin B. In again another embodiment, Vcontains a β-glucuronide that is specifically recognized byβ-glucuronidase present in the vicinity of or inside tumor cells. Inagain another embodiment the specifier is [O], yielding for example anitro group that can be reduced under hypoxic conditions or bynitroreductases. In another embodiment V is a nitro-(hetero)aromaticmoiety, for example nitrobenzyloxycarbonyl. After reduction of the nitrogroup or removal of the nitro-aromatic specifier, elimination of thespacers or spacer systems described in this invention leads to drugrelease. It can be understood that any specifier that is specificallycleaved following recognition by a disease-specific and/ororgan-specific and/or specifically targeted enzynme and/or receptor canbe incorporated into conjugates and prodrugs that contain the spacers orspacer systems claimed in this invention.

In one embodiment the invention relates to a compound wherein thespecifier V contains a tripeptide. Preferably the tripeptide is linkedvia its C-terminus to the self-eliminating multiple release spacer orspacer system. More preferably the C-terminal amino acid residue of thetripeptide is selected from arginine and lysine, the middle amino acidresidue of the tripeptide is selected from alanine, valine, leucine,isoleucine, methionine, phenylalanine, cyclohexylglycine, tryptophan andproline, and the N-terminal amino acid residue of the tripeptide isselected from a D-amino acid residue and a protected L-amino acidresidue including protected glycine.

In a further embodiment the specifier V is selected fromD-alanylphenylalanyllysine, D-valylleucyllysine, D-alanylleucyllysine,D-valylphenylalanyllysine, D-valyltryptophanyllysine andD-alanyltryptophanyllysine.

It should be noted that specifier V, either in the form of a di-, tri-oroligopeptide, or in any other form, may contain protective groups. Suchcompounds comprising protected specifier V may not, when contacted withfor instance specifier specific enzymes, release the leaving groups.However, when deprotected and suitably activated such compounds willrelease leaving groups and thus such compounds comprising protectedspecifier V also fall under the scope of this invention. In particularthe above can be envisaged in relation to the bifunctional ormonofunctional compoundsV—(W—)_(w)(X—)_(x)C((A—)_(a)S)_(c),V—(W—)_(w)(X—)_(x)C(D((A—)_(a)S)_(d))_(c),V—(W—)_(w)(X—)_(x)C(D(E((A—)_(a)S)_(e))_(d))_(c),V—(W—)_(w)(X—)_(x)C(D(E(F((A—)_(a)S)_(f))_(e))_(d))_(c)V—(W—)_(w)(X—)_(x)C((A—)_(a)Z)_(c),V—(W—)_(w)(X—)_(x)C(D((A—)_(a)Z)_(d))_(c),V—(W—)_(w)(X—)_(x)C(D(E((A—)_(a)Z)_(e))_(d))_(c), andV—(W—)_(w)(X—)_(x)C(D(E(F((A—)_(a)Z)_(f))_(e))_(d))_(c)mentioned earlier. Suitable protective groups for chemical functionalgroups, in particular for amino acids, are well known to the organicchemist and may for example be found in T. W. Greene, Protective Groupsin Organic Synthesis, John Wiley & Sons, New York, 1981.

In yet a further embodiment the invention relates to a compound whereinthe specifier V is an amino-terminal capped peptide covalently linkedvia the C-terminus to the self-eliminating multiple release spacer orspacer system. Preferably the specifier V is selected frombenzyloxycarbonylphenylalanyllysine, benzyloxycarbonylvalyllysine,D-phenylalanylphenylalanyllysine, benzyloxycarbonylvalylcitrulline,tert-butyloxycarbonylphenylalanyllysine,benzyloxycarbonylalanylarginylarginine,benzyloxycarbonylphenylalanyl-N-tosylarginine,2-aminoethylthiosuccinimidopropionylvalinylcitrulline,2-aminoethylthiosuccinimidopropionyllysylphenylalanyllysine,acetylphenylalanyllysine, andbenzyloxycarbonylphenylalanyl-O-benzoylthreonine.

The moiety Z is preferably a therapeutic or diagnostic moiety, but itcan also be a hydrogen or OH group or a reactive moiety. A H or OH groupor a reactive moiety may be accidentally introduced in a final conjugateduring its synthesis, in case final coupling of leaving groups to themultiple release spacer or spacer system does not proceed completely. Hor OH groups will not act as leaving group, but are not expected toinhibit elimination of leaving groups Z. Z can for instance be ananticancer drug, an antibiotic, an anti-inflammatory agent, or ananti-viral agent. Typically, the moiety Z is an anticancer drug.Preferably the anticancer drug is the hydroxyl containing etoposide,camptothecin, irinotecan (CPT-11), SN-38, topotecan,9-aminocamptothecin, 9-nitrocamptothecin, 10-hydroxycamptothecin, GG211,lurtotecan, combrestatin, paclitaxel, docetaxel, esperamycin,1,8-dihydroxy-bicyclo[7.3.1]trideca-4-ene-2,6-diyne-13-one, anguidine,doxorubicin, morpholine-doxorubicin, N-(5,5-diacetoxypentyl)doxorubicin, daunorubicin, epirubicin, idarubicin, mitoxantrone,vincristine, vinblastine, tallysomycin, bleomycin,4-bis(2-chloroethyl)aminophenol, 4-bis(2-fluoroethyl)aminophenol, andderivatives thereof.

The anticancer drug can also be the sulfhydryl containing esperamicin,6-mercaptopurine, or derivatives thereof. The anticancer drug can alsobe the carboxyl containing methotrexate, aminopterin, camptothecin(ring-opened form of the lactone), chlorambucil, melphalan, butyric acidand retinoic acid, and derivatives thereof. The anticancer drug can alsobe the aziridine amino containing or aromatic amino containing mitomycinC, nitomycin A, an anthracycline derivative containing an aminefunctionality with sufficient leaving group ability, mitoxantrone,9-aminocamptothecin, methotrexate, aminopterin, tallysomycin, bleomycin,actinomycin, N,N-bis(2-chloroethyl)-p-phenylenediaamine,N,N-bis(2-fluoroethyl)-p-phenylenediamine, deoxycytidine, cytosinearabinoside, gemcitabine, and derivatives thereof. The anticancer drugcan also be the aliphatic amino-containing daunorubicin, doxorubicin,epirubicin, idarubicin, N-(5,5-diacetoxypentyl)doxorubicin, ananthracycline, N⁸-acetyl spermidine,1-(2-chloroethyl)-1,2-dimethanesulfonyl hydrazine, or derivativesthereof.

In one embodiment Z is selected from paclitaxel, docetaxel, or aderivative thereof, which is coupled to the self-eliminating multiplerelease spacer or spacer system via its 2′-hydroxyl group. In a furtherembodiment Z is selected from camptothecin, irinotecan (CPT-11), SN-38,topotecan, 9-aminocamptothecin, 9-nitrocamptothecin,10-hydroxycamptothecin, GG211, lurtotecan, or a derivative thereof,which is coupled to the self-eliminating multiple release spacer orspacer system via its 20-hydroxyl group. In yet a further embodiment Zis selected from SN-38, topotecan, 10-hydroxycamptothecin, etoposide,4-bis(2-chloroethyl)aminophenol, 4-bis(2-fluoroethyl)aminophenol,combrestatin, or a derivative thereof, which is coupled to theself-eliminating multiple release spacer or spacer system via itsphenolic hydroxyl group. In a further embodiment Z is selected from9-amminocamptothecin, N,N-bis(2-chloroethyl)-p-phenylenediamine,N,N-bis(2-fluoroethyl)-p-phenylenediamine, or a derivative thereof,which is coupled to the self-eliminating multiple release spacer orspacer system via its aromatic primary amine group. In yet a furtherembodiment Z is selected from doxorubicin, daunorubicin, epirubicin,idarubicin, or a derivative thereof, which is coupled to theself-elimination multiple release spacer (system) via its aliphaticamino group, provided that at least one generation of the multiplerelease spacer system, preferably the highest generation, containsphenol- or thiophenol based spacers.

The moiety S is a reactive moiety that enables coupling of moieties Z tothe multiple release spacer (system). When the moiety S is connected viaa carbonyl group to the multiple release spacer or spacer system,suitable S moieties include, without limitation, N-succinimidyl-N-oxide,p-nitrophenoxide, pentafluorophenoxide, carboxylates, halides, andsulfonates. When the moiety S is directly connected to the methylenegroup of the multiple release spacer system, suitable S moietiesinclude, without limitation, halides and sulfonates. The moiety S mayalso be H or OH, in which case coupling to Z occurs by in situconversion of S to a reactive moiety which is then substituted by Z inthe same reaction mixture. The compounds of the invention that containtwo or more moieties S can be used to prepare the compounds of theinvention that contain two or more moieties Z. When the compounds of theinvention that contain two or more moieties S contain a specifier V thatalso contains a reactive moiety, the compounds have been namedbifunctional linkers. When the compounds of the invention that containtwo or more moieties S contain a specifier V that does not contain areactive moiety, the compounds are from hereon called “monofiuctionalspecifier-spacer conjugates”. Although in this invention spacers andspacer systems are described that are able to release multiple leavinggroups, it is obvious that monofunctional specifier-spacer conjugatesand bifunctional linkers can also be designed that contain singlerelease spacers and spacer systems, such as for example described in WO02/83180 and EP 1243276, which are incorporated by reference.

Preferred compounds according to the invention are those selected fromthe group consisting of

-   -   and salts thereof.

A compound consisting of an amino-terminal capped peptide covalentlylinked via one single p—NH—Ph—CH═C(CH₂OCO—)₂ spacer to two doxorubicinmolecules is excluded from the scope of this invention. Furthercompounds consisting of an amino-terminal capped peptide covalentlylinked via one single p—NH—Ph—CH═C(CH₂OCO—)₂ spacer to two anticancerdrug molecules may or may not be excluded from the scope of thisinvention.

When an enzyme needs to be quantitatively detected, incorporation of amultiple release spacer or spacer system into a compound or conjugatethat is enzymatically cleaved by said enzyme increases the sensitivityof such an assay as one enzymatic cleavage can liberate numerousdetectable molecules.

Detection or determination of an enzyme, for example a protease, in asample can be performed by incubating the sample with a compoundcontaining a multiple release spacer or spacer system according to thisinvention, which is a substrate for the (proteolytic) enzyme, andobserving (proteolytic) cleavage of said substrate. The phrase“determining an enzyme” means both qualitative analysis, i.e. detectingthe presence of the enzyme, determining whether it is present, andquantitative analysis, i.e. quantifying the enzyme, determining theenzyme activity present in the sample.

For many proteases chromogenic or fluorogenic peptide substrates havebeen devised which are often commercially available. In many cases, forexample in case of serine proteases, the enzymes do not recognize thesequence which is C-terminal to the hydrolyzed bond. This part can bereplaced by a chromogenic or fluorogenic leaving group likep-nitroaniline or β-naphtylamine. Such chromogenic or fluorogeniccompounds can serve as leaving group Z in compounds of the presentinvention. The multiple release spacer or spacer system may increase thesensitivity that can be obtained for detection and quantification ofphysiological concentrations of enzymes in biological fluids ortissue-extracts.

An enzyme can also be indirectly determined via its pro-enzymecontaining a recognition site, e.g., an activation site, cleavable bysaid enzyme to be determined. Cleavage of the pro-enzyme can in suchcase be detected by observing the resulting activity using a suitablesubstrate of the activated pro-enzyme, the substrate containing amultiple release spacer or spacer system of the present invention.

In one embodiment the invention relates to a diagnostic assay process inwhich a compound according to the invention is used.

In a further embodiment the invention relates to a process in which thepresence or amount of an enzyme is determined by using a compoundaccording to the invention.

In yet a further embodiment the invention relates to a process in whichthe presence or amount of a protease is determined by using a compoundaccording to the invention.

In a further embodiment the invention relates to a process in which thecompound according to the invention that is used comprises a substratefor said protease and leaving group Z is detected.

In yet a further embodiment the invention relates to a process in whichthe compound according to the invention that is used comprises asubstrate for an enzyme, which is the product of cleavage of itspro-enzyme precursor by said protease and leaving group Z is detected.

The specifier V may also comprise a polymer, such as for examplepoly[N-(2-hydroxy-propyl)methacrylamide] (poly-HPMA), poly-glutamic acid(poly-L-glutamic acid (PG)), or poly(ethylene) glycol (PEG), whichcauses accumulation of compounds of the invention in the vicinity of orinside the target cells, e.g. tumor cells, because of the EnhancedPermeability and Retention (EPR) effect. These polymers may as such beregarded as a targeting moiety. Two or more molecules of the inventionmay also be coupled to a central (core) monomeric or polymeric moleculeto obtain a larger structure that could accumulate as a result of theEPR effect, in addition to other mechanisms of accumulation. Suitablepolymers are for example poly[N-(2-hydroxy-propyl)methacrylamide](poly-HPMA), poly-glutamic acid (poly-L-glutamic acid (PG)), orpoly(ethylene) glycol (PEG). Thus the invention also relates to acomposite structure comprising two or more compounds according to theinvention, connected with a polymeric structure.

Structures of multiple release spacers are disclosed that can serve assuitable monomers to construct the multiple release dendrimeric prodrugsor (bio)conjugates. A suitable monomer is able to release 2 or moreleaving groups after activation. One herein disclosed structure of suchmultiple release monomer is depicted in FIG. 7. Triple release from thismonomer, as depicted in FIG. 7, is based on the principles of unbranched1,4- and 1,6-elimination spacers³⁰. After unmasking of the amine of 1(FIG. 7), 2 can undergo an electronic cascade reaction, after which thereactive non-aromatic species 3 is immediately trapped by a nucleophile,such as for example water, to regenerate the aromatic amine function in4. This process of self-elimination and trapping is repeated two moretimes, after which three leaving groups L—H have been liberated. Thesequence in which the three electronic cascade elimination reactionstake place as depicted in FIG. 7 was chosen randomly. In fact, the threefragmentations (two 1,4-eliminations and one 1,6-elimination) may occurin any random order. Employing n generations of this monomer, cascadedendrimers can potentially be constructed that contain 3^(n) end groups.

FIG. 8 depicts the principle of drug release from a double releasespacer. Self-elimination of this double release spacer is based on1,8-elimination. Deprotection of the amino group in 7, affording 8, setsthe stage for 2 1,8-elinination reactions releasing 2 leaving groupsL—H.

An amine or hydroxylamine can be generated from a masked amine throughreduction of a nitro function to the corresponding hydroxylamine oramine function. Both the hydroxylamine and the amine derivatives induceself-elimination of the spacer.

To deliver proof of principle for multiple release of leaving groupsfrom conjugates and prodrugs containing a multiple release spacer orspacer system as disclosed herein, compounds were synthezised thatcontain multiple leaving groups coupled as end groups to one or twogenerations of multiple release spacers. These compounds were to releasethe leaving groups upon reduction of the nitro group attached to thearomatic ring at the center of the multiple release linker or linkersystem. Reduction of the nitro group to the corresponding hydroxylamineor amine should trigger the multiple release cascade and simultaneouslyliberate all leaving groups. Alternatively, removal of an Alocprotecting group to unmask the amine can trigger self-elimination. Inaddition to serving as compounds to deliver proof of principle, thesynthesized nitroaromatic compounds that contain antitumor drugs, suchas paclitaxel, as leaving groups can be considered for application inselective drug targeting to hypoxic areas in tumors, where the nitrogroup will be reduced. Alternatively, these compounds can be used asconjugates and prodrugs that are activated by nitroreductases which aretargeted to tumor cells through one of the directed enzyme prodrugtherapy (DEPT) approaches. The synthesis of these compounds isdisclosed.

The triple release building blocks nitrotriol 11 (FIG. 9) and aminotriol16 (FIG. 11) can be synthesized following a number of routes. Severalapproaches to obtain the corresponding amino-tri-methylester oramino-tri-carboxylic acid have been reported starting from for examplenitromesitylene³¹.

Synthesis of the double release building block nitrodiol 20 (FIG. 12)has been reported in the literature³². The double release building blockaininodiol 23 (FIG. 13) can be synthesized by reduction of the nitrogroup of 2-p-nitrobenzylidene propane-1,3-diol 20.

Proof of principle of triple release should be delivered with amonomeric model compound with a structure similar to 1 (FIG. 7). Toobtain such a compound, nitrotriol 11 was activated with excessp-nitrophenyl chloroformate in the presence of diisopropylethylamine(DIPEA) and pyridine to yield triply activated compound 12 (FIG. 9).³³Triply activated 12 was subsequently reacted with benzyl alcohol toyield model compound 13, or with phenethyl alcohol to yield modelcompound 14. Triply activated 12 was also reacted with benzylamine toobtain model compound 15 (FIG. 10). In order to obtain model compound 19(FIG. 11), aminotriol 16 was N-protected with an Aloc group to yield 17,subsequently triply activated with p-nitrophenyl chloroformate to yield18, and coupled with propylamine.

Proof of principle for the fact that the dual release spacer with astructure similar to 10 (FIG. 8) can indeed release both leaving groupsis also disclosed in this invention. For this purpose, nitrodiol 20 wasdoubly activated with excess p-nitrophenyl chloroformate in the presenceof diisopropylethylamine (DIPEA) and pyridine to yield doubly activatedcompound 21 (FIG. 12). Doubly activated 21 was subsequently reacted withpaclitaxel to yield the desired model compound 22.

In addition to delivering proof of principle for double release from aspacer with a structure similar to 10 (FIG. 8), a model compound wassynthesized for delivering proof of principle for release of 4 leavinggroups from a multiple release prodrug or (bio)conjugate containing 2generations of double release spacers. For this purpose, two moleculesof aminodiol 23 were coupled to doubly activated 21 using HOBt as acatalyst to yield tetraol 24 (FIG. 13). Tetraol 24 was quadruplyactivated with excess p-nitrophenyl chloroformate in the presence ofdiisopropylethylamine (DIPEA) and pyridine to yield quadruply activatedcompound 25. Quadruply activated 25 was subsequently reacted withpaclitaxel to yield the desired model compound 26.

Doubly activated 21 was also reacted with benzylamine to yield modelcompound 27 (FIG. 14). To synthesize additional model compounds, doublyactivated 21 was also reacted with the single release 1,6-eliminationspacer to yield 28 (FIG. 15), which was subsequently activated usingp-nitrophenyl chloroformate to yield 29. Doubly activated 29 was reactedwith p-methoxybenzylamine, p-chlorobenzylamine, or phenethyl alcohol, toyield model compounds 30, 31, and 32, respectively (FIG. 16).

To obtain a reference compound for demonstration of single1,6-elimination, p-nitrobenzyl chloroformate 33 was reacted withbenzylamine to yield model compound 34 (FIG. 17).

Two other reference compounds were also prepared (FIG. 18). Dibenzylcarbonate 35 and 2′-O-cinnamyloxycarbonylpaclitaxel 36 were synthesizedas reference compounds to demonstrate that these compounds remain intactupon treatment with conditions that are necessary for reduction of thenitro function of the model compounds described above.

Although the conjugates, test compounds, and reference compounds arebased on aniline-based spacers, it is obvious to one skilled in the artthat similar compounds containing one or more phenol- orthiophenol-based spacers can be prepared analogously.

In order to deliver proof of principle for release of all three leavinggroups, 13 and 14 were unmasked by reduction of the nitro group usingzinc in the presence of acetic acid (FIG. 19). Within one hour thinlayer chromatography indicated complete disappearance of startingcompound and formation of a substantial amount of benzyl alcohol orphenethyl alcohol. NMR spectra of the products indicated completeformation of free benzyl alcohol or phenethyl alcohol, no signals frombenzyl carbonate or phenethyl carbonate protons being present.

When the nitro function of model compound 15 was reduced (FIG. 20), thinlayer chromatography indicated formation of a single product. However,formation of benzylamine was not or hardly observed. According to NMR,maximally 33 percent of benzylamine was released from 15.

Treatment of the model compound 19 (FIG. 21) withpalladiumtetrakistriphenylphosphine and morpholine resulted indisappearance of starting compound within one hour. However, again nocomplete release of propylamine was observed.

In order to deliver proof of principle for release of both paclitaxelleaving groups, 22 was unmasked by reduction of the nitro group usingzinc in the presence of acetic acid (FIG. 22). Within 30 minutes thinlayer chromatography indicated complete disappearance of startingcompound and formation of a substantial amount of paclitaxel. An NMRspectrum of the product indicated complete formation of free paclitaxel,no signals from paclitaxel-2′-carbonate protons were present.

A similar reduction of the nitro group of the model compound 26,containing 2 generations of multiple release spacers and containing 4paclitaxel units, led to complete disappearance of starting materialwithin 30 minutes and a substantial amount of paclitaxel was formedaccording to thin layer chromatography (FIG. 23). Also in this case, anNMR spectrum of the product indicated complete formation of freepaclitaxel.

Treatment of model compound 27, containing benzylarnine as leavinggroups (FIG. 24), with zinc showed disappearance of starting compound.However, again no complete release of benzylamine was observed.

Also model compounds 30 and 31, containing aliphatic amine leavinggroups (p-methoxybenzylamine or p-chlorobenzylamine), were reduced. Inboth cases, within 30 min, the starting compound was converted into asingle product, however, again no free amine was formed.

When the phenethyl alcohol leaving group containing compound 32 wasreduced under the same conditions, the product showed to be completelyconverted into free phenethyl alcohol, as observed by ¹H-NMR..

To verify whether the single release single release 1,6-eliminationspacer self-eliminates when the leaving group is an aliphatic amine,compound 34 was reduced. NMR indicated complete release of benzylamine.

The reference compounds 35 and 36 were also treated with zinc and aceticacid (FIG. 28). According to ¹H-NMR both compounds did not show anydegradation, which demonstrates that the liberation of leaving groupsfrom the nitro-containing model compounds is not due to unwanted sidereactions.

From some reduction products of aliphatic amine leaving group containingmodel compounds, NMR or thin layer chromatography showed in some casesafter 1-3 months following nitro reduction or Aloc deprotection, thatpercentages of liberated leaving group remained unchanged. In each case,no more than one equivalent of aliphatic amine was released. Both theherein disclosed double and triple release aniline-based multiplerelease spacers are not able to release more than one leaving group, ifthe leaving group is an aliphatic amine. The multiple release structuresas depicted in FIGS. 7 and 8 have been shown in this invention topossess multiple release characteristics when the leaving group is anaromatic amine or an oxygen (hydroxyl). Apparently, the extent ofelectron-withdrawing capacity of the leaving groups deterrnines whethermore than one leaving group can be liberated from the herein disclosedspacers. When the multiple release spacer is a phenol- orthiophenol-based multiple release spacer or when the multiple releasespacer is coupled to single release phenol- or thiophenol-based spacers,aliphatic amines are also suitable leaving groups and release of allaliphatic amine leaving groups does occur. Aliphatic alcohols prove tobe suitable leaving groups (e.g., fromH₂N—C₆H₄—CH═C(CH₂—O—CO—2′—O-paclitaxel)₂ two molecules of paclitaxel arereleased) and therefore release of phenol-based spacers, to which analiphatic amine leaving group is connected, from a multiple releasespacer (e.g., H₂N—C₆H₄—CH═C(CH₂—O—C(O)O—C₆H₄—CH₂OC(O)NR¹R²)₂) occurs byvirtue of their increased leaving capability with respect to aliphaticalcohols. As elimination of aliphatic amines from hydroxybenzyl-basedspacer-aliphatic amine conjugates is known¹⁰, release of aliphaticamines from spacer systems in which the aliphatic amine is directlyconnected to a phenol-based spacer takes place as well.

Upon removal or transformation of the specifier V in the compounds orprodrugs disclosed in this invention, thus upon unmasking of B of thebranched spacer or spacer system coupled to V, under physiologicalconditions, the reactive intemiediate(s) formed after self-eliminationis (are) likely to be trapped by water, thereby regenerating moleculeslike aminodiol 23 and/or aminotriol 16. When a multiple releaseconjugate or prodrug constructed with one of the monomers disclosed inthis invention is used for drug delivery purposes, the degradationproducts of the prodrug or (bio)conjugate after the multipleself-elimination cascade must be non-toxic (except from drugs to bereleased in tumor targeting approaches). Therefore, we have tested theaminotriol monomer 16 and aminodiol monomer 23 for their cytotoxicity ina panel of seven weul-characterized human tumor cell lines. Bothcompounds turned out to be non-toxic (see example 33).

The total attainable number of end groups (e.g., drug moieties) perdendrimeric prodrug or conjugate may be restricted by steric conditions.When incorporation of multiple large bioactive end groups is desired, itis likely that only several generations of multiple release monomericspacers can be used. The number of end groups that can be incorporatedmay be increased by addition of additional unbranched self-eliminationspacer(s) (systems) to a generation of multiple release spacers or tothe highest generation of multiple release spacer(s) before leavinggroups Z in order to enlarge the outer sphere surface, thereby enablingattachment of more end groups.

The electronic cascade self-elimination process of 4-aminobenzyl alcohol1,6-elimination spacers is known to proceed with a short half-live. Thisis an important factor in drug delivery applications of self-eliminationspacers, as fast drug release after site-specific activation isrequired. When a slower release of leaving groups is desired, the spacermonomer may be tuned such that the spacer elimination process isretarded. This can be of use when the functional multiple releasespacers or spacer systems of this invention are used for controlledrelease purposes.

Unbranched and branched monomeric aminobenzyloxycarbonyl and relatedelectronic cascade spacers or spacer systems can be coupled to a nextgeneration of monomeric spacers via carbamate bonds, which arepresumably more stable under physiological conditions than ester orcarbonate bonds³⁴.

In the present invention, the synthesis and application of new multiplerelease spacers and spacer systems is described. In one embodiment, thedual or triple release monomer(s) employed in the multiple releaseprodrug or (bio)conjugate release the end groups (drugs) through severalconsecutive 1,4-, 1,6-, or 1,8-eliminations. Proof of principle ofdouble or triple self-elimination was delivered upon chemical reductionof the nitrobenzyl or nitrocinnamyl derivatives containing benzylalcohol, phenethyl alcohol, or paclitaxel as leaving groups (FIGS. 19,22, 23, 26). Complete release was observed by thin layer chromatography,and was unambiguously confirmed by NMR. Also the compound containing 2generations of double release monomers and 4 paclitaxel moieties wasshown to release all end groups following reduction of the nitro group(FIG. 23).

Obviously, unbranched self-elimination spacers may be incorporated inbetween the specifier and the multiple release spacers. This may bebeneficial for the enzymatic removal or transformation of the specifier.A branched multiple release moiety directly coupled to the specifier mayincrease steric crowding around the specifier-spacer bond to be cleaved;additional single release self-elimination spacers may provide asolution here.

Thus, for efficient removal or transformation of the specifier, in somecases it is particularly preferred to incorporate one or more singlerelease self-elimination spacers in between specifier and one or moregeneration(s) of a multiple release spacer. This means that in compoundsof formulaV—(W—)_(w)(X—)_(x)C((A—)_(a)Z)_(c),V—(W—)_(w)(X—)_(x)C(D((A—)_(a)Z)_(d))_(c),V—(W—)_(w)(X—)_(x)C(D(E((A—)_(a)Z)_(e))_(d))_(c), andV—(W—)_(w)(X—)_(x)C(D(E(F((A—)_(a)Z)_(f))_(e))_(d))_(c),w+x>0.

In addition, single release self-elimination spacers may be added to thehighest generation of the multiple release spacer(s) before leavinggroups Z. This may be beneficial for enhanced release of Z, depending onthe chemical nature of the function with which Z, preferably a drug, isattached to the spacer. Incorporation of single release self-eliminationspacers before Z (for example drug moieties) may be beneficial for thestability of the final product. If, for example, a self-eliminationω-amino aminocarbonyl cyclization spacer is incorporated in between thesingle release or multiple release electronic cascade spacer and aleaving group that is connected through its hydroxyl group, thestability of the final product may be increased when used underphysiological conditions. Furthermore, when only aniline-based multiplerelease spacers are used, addition of single release phenol- orthiophenol-based spacers to a generation of multiple release spacers or,preferably, to the highest generation of multiple release spacer(s)before leaving groups Z enables the release of two or more leavinggroups Z that are coupled with their aliphatic amino group to themultiple release spacer system. When only aniline-based multiple releasespacers are used and no single release phenol- or thiophenol-basedspacers are incorporated into the multiple release spacer system towhich leaving groups Z are coupled with their aliphatic amino group only1 Z group will be released.

In yet another aspect the invention relates to the use of any of thecompounds defined above for the manufacture of a pharmaceuticalpreparation for the treatment of a mammal being in need thereof. Theinvention also relates to methods of treating a mammal being in needthereof, whereby the method comprises the administration of apharmaceutical composition to the mammal in a therapeutically effectivedose.

In a further aspect the invention relates to a process for preparing apharmaceutical composition containing a compound as defined above, toprovide a solid or a liquid formulation for administration orally,topically or by injection. Such a process at least comprises the step ofmixing the compound with a pharmaceutically acceptable carrier.

The invention also relates to pharmaceutical compositions comprising thecompounds of the invention as defined above. The compounds of theinvention may be administered in purified form together with apharmaceutical carrier as a pharmaceutical composition. The preferredform depends on the intended mode of administration and therapeutic ordiagnostic application. The pharmaceutical carrier can be anycompatible, nontoxic substance suitable to deliver the compounds of theinvention to the patient. Pharmaceutically acceptable carriers are wellknown in the art and include, for example, aqueous solutions such as(sterile) water or physiologically buffered saline or other solvents orvehicles such as glycols, glycerol, oils such as olive oil or injectableorganic esters, alcohol, fats, waxes, and inert solids may be used asthe carrier. A pharmaceutically acceptable carrier may further containphysiologically acceptable compounds that act, e.g. to stabilize or toincrease the absorption of the compounds of the invention. Suchphysiologically acceptable compounds include, for example,carbohydrates, such as glucose, sucrose or dextrans, antioxidants, suchas ascorbic acid or glutathione, chelating agents, low molecular weightproteins or other stabilizers or excipients. One skilled in the artwould know that the choice of a pharmaceutically acceptable carrier,including a physiologically acceptable compound, depends, for example,on the route of administration of the composition. Pharmaceuticallyacceptable adjuvants, buffering agents, dispersing agents, and the like,may also be incorporated into the pharmaceutical compositions.

For oral administration, the active ingredient can be administered insolid dosage forms, such as capsules, tablets, and powders, or in liquiddosage forms, such as elixirs, syrups, and suspensions. Activecomponent(s) can be encapsulated in gelatin capsules together withinactive ingredients and powdered carriers, such as glucose, lactose,sucrose, mannitol, starch, cellulose or cellulose derivatives, magnesiumstearate, stearic acid, sodium saccharin, talcum, magnesium carbonateand the like. Examples of additional inactive ingredients that may beadded to provide desirable color, taste, stability, buffering capacity,dispersion or other known desirable features are red iron oxide, silicagel, sodium lauryl sulfate, titanium dioxide, edible white ink and thelike. Similar diluents can be used to make compressed tablets. Bothtablets and capsules can be manufactured as sustained release productsto provide for continuous release of medication over a period of hours.Compressed tablets can be sugar coated or film coated to mask anyunpleasant taste and protect the tablet fiom the atmosphere, orenteric-coated for selective disintegration in the gastrointestinaltract. Liquid dosage forms for oral administration can contain coloringand flavoring to increase patient acceptance.

The compounds of the invention are however preferably administeredparenterally. Preparations of the compounds of the invention forparenteral administration must be sterile. Sterilization is readilyaccomplished by filtration through sterile filtration membranes,optionally prior to or following lyophilization and reconstitution. Theparenteral route for administration of compounds of the invention is inaccord with known methods, e.g. injection or infusion by intravenous,intraperitoneal, intramuscular, intraarterial, or intralesional routes.The compounds of the invention may be administered continuously byinfusion or by bolus injection. A typical composition for intravenousinfusion could be made up to contain 100 to 500 ml of sterile 0.9% NaClor 5% glucose optionally supplemented with a 20% albumin solution and 1mg to 10 g of the compound of the invention, depending on the particulartype of compound of the invention and its required dosing regime.Methods for preparing parenterally admninistrable compositions are wellknown in the art and described in more detail in various sources,including, for example, Remington's Pharmaceutical Science³⁵.

The invention also relates to compounds as defined above, wherein thespecifier V is removed or transformed by an enzyme that is transportedto the vicinity of or inside target cells or target tissue viaantibody-directed enzyme prodrug therapy (ADEPT)¹, polymer-directedenzyme prodrug therapy (PDEPT) or macromolecular-directed enzyme prodrugtherapy (MDEPT)², virus-directed enzyme prodrug therapy (VDEPT)³ orgene-directed enzyme prodrug therapy (GDEPT)⁴.

The invention is further exemplified by the following Examples. Theseexamples are for illustrative purposes and are not intended to limit thescope of the invention.

EXAMPLES Example 1

Activation of nitrotriol 11 to give triply activated 12 (see FIG. 9).100 mg (0.47 nmmol) of nitrotriol 11 was dissolved in 6 mL of dry THFunder an argon atmosphere. The solution was added dropwise to a solutionof 4-nitrophenyl chloroformate (1.42 g, 15 equiv) and pyridine (569 μL,15 equiv) in 5 mL dry THF. The reaction mixture was stirred for 1 h,dichloromethane was added, and the organic layer was washed with 10%citric acid and brine. The organic layer was dried over anhydrous sodiumsulfate and evaporated to dryness. The product was purified by means ofcolumn chromatography (EtOAc/heptane 3/5) to afford 139 mg (42%) oftriply activated 12. ¹H-NMR (300 MHz, CDCl₃) δ 5.41 (s, 2H, CH₂), 5.47(s, 4H, CH₂), 7.37-7.42 (m, 6H, aromatic), 7.76 (s, 2H, aromatic),8.26-8.30 (m, 6H, aromatic) ppm; FAB-MS m/e 709 (M+H)⁺, 731 (M+Na)⁺;Anal. (C₃₀H₂₀O₁₇N₄) calculated C 50.86%, H 2.85%, N 7.91%, measured C51.09%, H 3.13%, N 7.54%.

Example 2

Coupling of benzyl alcohol to triply activated 12 to give 13 (see FIG.9). Triply activated 12 (29.5 mg, 0.042 mmol) was dissolved in drydichloromethane. Benzyl alcohol (13 μL, 3.1 equiv) and DMAP (17 mg, 3.3equiv) were added and the reaction mixture was stirred for 64 h.Dichloromethane was added and the organic layer was washed withsaturated sodium bicarbonate, 10% citric acid, and brine, and dried oversodium sulfate. The product was purified by means of columnchromatography (EtOAc/heptane 3/5) to afford 15 mg (59%) of desiredproduct 13. ¹H-NMR (300 MHz, CDCl₃) δ 5.15-5.18 (m, 8H, benzylic), 5.27(s, 4H, benzylic), 7.33-7.39 (m, 15H, aromatic benzyl alcohol), 7.52 (s,2H, aromatic spacer) ppm.

Example 3

Coupling of phenethyl alcohol to triply activated 12 to give 14 (seeFIG. 9). Triply activated 12 (27 mg, 0.038 mmol) was dissolved in drydichloromethane and the solution was cooled to 0° C. Phenethyl alcohol(16 μL, 3.5 equiv) and DMAP (16 mg, 3.5 equiv) were added and thereaction mixture was allowed to reach Rt and was stirred for 40 h.Dichloromethane was added and the organic layer was washed withsaturated sodium bicarbonate, 10% citric acid, and brine, and dried oversodium sulfate. The product was purified by means of columnchromatography (EtOAc/heptane 1/1) to afford 14 mg (57%) of desiredproduct 14. ¹H-NMR (300 MHz, DMSO-D₆/CDCl₃/CD₃OD) δ 2.91 (m, 6H, CH₂CH₂Ph), 4.30 (m, 6H, CH ₂CH₂Ph), 5.18 (m, 6H, OCH ₂Ph), 7.14-7.28 (m, 15H,aromatic), 7.58 (s, 2H, aromatic) ppm; ESI-MS m/e 680 (M+Na)⁺.

Example 4

Coupling of benzylamine to triply activated 12 to give 15 (see FIG. 10).To a solution of 12 (523 mg, 0.738 mmol) in dichloromethane were addedbenzylamine (403 μL, 3.69 mmol) and Et₃N (514 μL, 3.69 mmol). Thereaction mixture was stirred at room temperature for 4 h.Dichlorormethane was added and the resulting mixture was washed with 10%citric acid, saturated sodium bicarbonate and brine, dried withanhydrous sodium sulfate, and concentrated under reduced pressure.Column chromatography (CH₂Cl₂/MeOH 25/1) gave 15 (406 mg, 90%) as asolid. ¹H-NMR (300 MHz, CDCl₃) δ 4.31-4.36 (m, 6H, benzylicbenzylamine), 5.13 (s, 2H, benzylic spacer), 5.20 (s, 4H, benzylicspacer), 7.23-7.34 (m, 17 H, aromatic) ppm.

Example 5

Protection of aminotriol 16 to give allylN-[2,4,6-tri(hydroxymethyl)phenyl] carbamate 17 (see FIG. 11). To asolution of 1.085 g of 16 (5.92 mmol) in 20 mL dry THF under an argonatmosphere were added 1.48 mL (Aloc)₂O (1.5 equiv) and 200 mg HOBt (0.25equiv). The reaction mixture was stirred for 72 hours at roomtemperature. THF was evaporated and the product was purified by means ofcolumn chromatography (CH₂Cl₂/MeOH 6/1) which afforded 872 mg (55%) ofthe desired compound 17. Mp 87° C.; ¹H-NMR (300 MHz, CDCL₃/CD₃OD) δ4.58-4.66 (m, 8H, 3×CH₂ and 2 Aloc), 5.25-5.41 (m, 2H, Aloc), 5.86-6.11(m, 1H, Aloc), 7.35 (s, 2H, aromatic) ppm; MS (EI) m/e 249 (M−H₂O)⁺;Anal. (C₁₃H₁₇NO₅·7/8H₂O) calculated C 55.17%, H 6.68%, N 4.95%, measuredC 55.16%, H 6.16%, N 4.81%.

Example 6

Activation of triol 17 to give triply activated tricarbonate 18 (seeFIG. 11). A solution of 837 mg of triol 17 (3.13 mmol) in dry THF wasadded drop wise under an argon atmosphere to a solution of 9.47 g4-nitrophenyl chloroformate (15 equiv) and 3.8 mL pyridine (15 equiv) indry THF. The reaction mixture was stirred for 5 hours and THF wasevaporated. CH₂Cl₂ was added and the organic layer was washed with 10%aqueous citric acid and brine. The organic layer was dried overanhydrous Na₂SO₄ and evaporated. The product was purified by means ofcolumn chromatography (EtOAc/heptane 2/5) to afford 1.546 g (65%) oftricarbonate 18. Mp 46° C.; ¹H-NMR (300 MHz, CDCL₃) δ 4.68 (d, 2H, J=5.7Hz, Aloc), 5.22-5.37 (m, 8H, 3×CH₂ and 2 Aloc), 5.89-6.07 (m, 1H, Aloc),7.35-7.42 (m, 6H, aromatic), 7.67 (s, 2H, aromatic), 8.26-8.30 (m, 6H,aromatic) ppm; MS (EI) m/e 763 (M+H)⁺, 785 (M+Na)⁺; Anal.(C₃₄H₂₆N₄O₁₇·3H₂O) calculated C 50.00%, H 3.95%, N 6.86%, measured C49.99%, H 3.33%, N 6.44%.

Example 7

Coupling of propylamine to triply activated 18 to give triscarbamate 19(see FIG. 11). To a solution of 18 (313 mg, 0.410 mmol) indichloromethane were added propylamine (169 μL, 2.05 mmol) and Et₃N (286μL, 2.05 mmol). The reaction mixture was stirred at room temperature for16 h. Dichloromethane was added and the resulting mixture was washedwith 10% citric acid, saturated sodium bicarbonate and brine, dried withanhydrous sodium sulfate, and concentrated under reduced pressure.Column chromatography (CH₂Cl₂/MeOH 20/1) gave 19 (158 mg, 74%) as asolid. ¹H-NMR (300 MHz, CDCl₃) δ 0.87-0.94 (m, 9H, Me), 1.43-1.56 (m,6H, CH₂CH ₂CH₃), 3.07-3.15 (m, 6H, CH ₂CH₂CH₃), 4.63 (d, 2H, Aloc),5.01-5.17 (m, 8H, 2H Aloc and 6H O—CH₂), 5.95 (m, 1H, Aloc), 7.32 (bs,2H, aromatic) ppm.

Example 8

Activation of nitrodiol 20 to give doubly activated 21 (see FIG.12).:Nitrodiol 20 (130 mg, 0.621 mmol) was dissolved in 3 mL of dry THFunder an argon atmosphere and the solution was cooled to 0° C.Diisopropylethylamine (DIPEA) (865 μL, 8 equiv), 4-nitrophenylchloroformate (751 mg, 6 equiv), and pyridine (25 μL, 0.5 equiv) wereadded to the solution. The reaction mixture was allowed to reach roomtemperature (Rt) and stirred for 16 h. Dichloromethane was added and theorganic layer was washed with water, saturated sodium bicarbonate, andwater. The organic layer was dried over anhydrous sodium sulfate andevaporated to dryness. The product was purified by means of columnchromatography (EtOAc/heptane 2/5) to afford 280 mg (84%) of doublyactivated 21. ¹H-NMR (300 MHz, CDCl₃) δ 5.04 (s, 2H, CH₂), 5.08 (s, 2H,CH₂), 7.12 (s, 1H, vinylic), 7.38 (d, 2H, J=3.9 Hz, aromatic), 7.41 (d,2H, J=3.9 Hz, aromatic), 7.51 (d, 2H, J=8.3 Hz, aromatic), 8.25-8.30 (m,6H, aromatic) ppm; Anal. (C₂₄H₁₇N₃O₁₂) calculated C 53.44%, H 3.18%, N7.79%, measured C 53.68%, H 3.44%, N 7.31%.

Example 9

Coupling of paclitaxel to doubly activated 21 to give 22 (see FIG. 12).Doubly activated 21 (40 mg, 0.075 mmol) was dissolved in 1 mL of drydichloromethane and the solution was cooled to 0° C. Paclitaxel (128 mg,2 equiv) and 4-(dimethylamino)pyridine (DMAP) (20 mg, 2.2 equiv) wereadded and the reaction mixture was allowed to reach Rt and was stirredfor 16 h. Dichloromethane was added and the organic layer was washedwith saturated sodium bicarbonate, 0.5 N potassium bisulfate, and brine,and dried over sodium sulfate. The product was purified by means ofcolumn chromatography (EtOAc) to afford 118 mg (80%) of desired product22. ¹H-NMR (300 MHz, CDCl₃) δ 1.14 (s, 6H, 17), 1.26 (s, 6H, 16), 1.68(s, 6H, 19), 1.91 (bs, 6H, 18), 2.21 (s, 3H, 10-acetyl), 2.22 (s, 3H,10-acetyl), 2.46 (s, 6H, 4-acetyl), 3.80 (bd, 2H, 3), 4.19 (d, 2H, 20b),4.31 (d, 2H, 20a), 4.43 (m, 2H, 7), 4.76 (dd, 2H, CH₂-cinnanyl), 4.85(s, 2H, CH₂-cinnamyl), 4.95 (d, 2H, 5), 5.46 (d, 1H, J=3.0 Hz, 2′), 5.49(d, 1H, J=3.1 Hz, 2′), 5.68 (d, 2H, J=7.2 Hz, 2), 6.05 (m, 2H, 3′),6.20-6.35 (m, 4H, 10 and 13), 6.92 (s, 1H, vinylic cinnamyl), 7.30-7.65(m, 24H, aromatic), 7.71 (m, 4H, aromatic N-Bz), 8.12-8.16 (m, 6H,aromatic, 4H 2-Bz and 2H cinnamyl) ppm; ESI-MS m/e 1993 (M+Na)⁺; Anal.(C₁₀₆H₁₀₉N₃O₃₄) calculated C 64.66%, H 5.58%, N 2.13%, measured C65.02%, H 6.07%, N 1.72%.

Example 10

Coupling of aminodiol 23 to doubly activated 21 to give tetraol 24 (seeFIG. 13). Doubly activated 21 (154 mg, 0.285 mmol) was dissolved in 3 mLof dry dimethylformamide (DMF) and aminodiol 23 (113 mg, 2.2 equiv),DIPEA (99 μL, 2 equiv), and a catalytic amount of hydroxybenzotriazolemonohydrate (HOBt) (15 mg, 0.39 equiv) were added. The reaction mixturewas stirred for 16 h. HOBt (8 mg, 0.2 equiv) was added and the reactionmixture was stirred for another 64 h. 10% 2-propanol in EtOAc was addedand the organic layer was washed with water and dried over sodiumsulfate. The crude product was purified by means of columnchromatography (dichloromethane/MeOH 6/1) to yield tetraol 24 (72 mg,41%). ¹H-NMR (300 MHz, CDCl₃/CD₃OD) δ 4.31 (m, 8H, CH ₂OH), 4.91 (s, 2H,CH ₂OCO), 4.95 (s, 2H, CH ₂OCO), 6.62 (s, 2H, vinylic), 6.98 (s, 1H,vinylic), 7.24 (d, 2H, J=8.6 Hz, aromatic), 7.24 (d, 2H, J=8.6 Hz,aromatic), 7.40 (m, 4H, aromatic), 7.56 (d, 2H, J=8.5 Hz, aromatic),8.26 (d, 2H, J=8.8 Hz, aromatic) ppm; FAB-MS m/e 643 (M+Na)⁺; Anal.(C₃₂H₃₃N₃O₁₀) calculated C 62.03%, H 5.37%, N 6.78%, measured C 62.62%,H 5.71%, N 6.18%.

Example 11

Activation of tetraol 24 to give quadruply activated 25 (see FIG. 13).Tetraol 24 (58 mg, 0.094 mmol) was dissolved in dry THF and the solutionwas cooled to 0° C. DIPEA (261 μL, 16 equiv), 4-nitrophenylchloroformate (226 mg, 12 equiv), and pyridine (8 μL, 16 equiv) wereadded to the solution. The reaction mixture was allowed to reach Rt andwas stirred for 16 h. Dry dichloromethane was added and the reactionmixture was stirred for another 24 h. Dichloromethane was added and theorganic layer was washed with saturated sodium bicarbonate, 10% citricacid, and brine. The organic layer was dried over anhydrous sodiumsulfate and evaporated to dryness. The product was purified by means ofcolumn chromatography (EtOAc/heptane 5/4) to afford 65 mg (54%) ofquadruply activated 25. ¹H-NMR (300 MHz, CDCl₃) δ 4.93 (s, 2H, CH₂OCONH), 4.95 (s, 2H, CH ₂OCONH), 5.03 (s, 4H, CH ₂OCOO), 5.07 (s, 4H,CH ₂OCOO), 6.80-7.02 (m, 3H, vinylic), 7.28-7.50 (m, 18H, aromatic),8.23-8.29 (m, 10H, aromatic) ppm; ESI-MS m/e 1302 (M+Na)⁺; Anal.(C₆₀H₄₅N₇O₂₆) calculated C 56.30%, H 3.54%, N 7.66%, measured C 56.58%,H 3.74%, N 7.37%.

Example 12

Coupling of paclitaxel to quadruply activated 25 to give 26 (see FIG.13). Quadruply activated 25 (13 mg, 0.010 mmol) was dissolved in 1 mL ofdry dichloromethane and the solution was cooled to 0° C. Paclitaxel (35mg, 4 equiv) and DMAP (5.5 mg, 4.4 equiv) were added and the reactionmixture was allowed to reach Rt and was stirred for 16 h.Dichloromethane was added and the organic layer was washed withsaturated sodium bicarbonate, 0.5 N potassium bisulfate, and brine, anddried over sodium sulfate. The product was purified by means of columnchromatography (EtOAc) to afford 27 mg (64%) of desired product 26.¹H-NMR (300 MHz, CDCl₃) δ 1.15 (m, 12H, 17), 1.26 (m, 12H, 16), 1.68(bs, 12H, 19), 1.89 (m, 12H, 18), 2.17 (m, 12H, 10-acetyl), 2.42 (m,12H, 4-acetyl), 3.80 (m, 4H, 3), 4.20 (ni, 4H, 20b), 4.29 (m, 4H, 20a),4.40 (m, 4H, 7), 4.75-5.00 (m, 16H, 4H 5 and 12H CH₂-cinnamyl), 5.47 (m,4H, 2′), 5.65 (m, 4H, 2), 5.97 (m, 4H, 3′), 6.13-6.38 (m, 8H, 10 and13), 6.76-6.91 (m, 3H, vinylic cinnamyl), 7.23-7.65 (m, H, aromatic),7.71 (d, 8H, aromatic N-Bz), 8.13 (m, 8H, aromatic 2-Bz), 8.23 (d, 2H,aromatic cinnamyl) ppm; ESI-MS m/e 4157 (M+H₂O)⁺.

Example 13

Coupling of benzylamine to doubly activated 21 to give 27 (see FIG. 14).To a solution of 21 (50 mg, 92.7 μmol) in dichloromethane (2 mL) wereadded benzylamine (51 μL, 0.464 mmol) and Et₃N (65 μL, 0.464 mmol). Thereaction mixture was stirred at room temperature for 16 h.Dichloromethane was added and the resulting mixture was washed with 10%citric acid, saturated bicarbonate and brine, dried with anhydroussodium sulfate, and concentrated under reduced pressure. Columnchromatography (CH₂Cl₂/MeOH 40/1) gave 27 (38 mg, 79.9 μmol, 86%) as asolid. ¹H-NMR (300 MHz, CDCl₃) δ 4.33-4.39 (m, 4H, benzylic), 4.79 (s,2H, spacer), 4.82 (s, 2H, spacer), 6.78 (s, 1H, alkene), 7.25-7.35 (m,12H, aromatic), 7.42 (d, 2H, aromatic spacer), 8.18 (d, 2H, aromaticspacer) ppm.

Example 14

Coupling of p-aminobenzyl alcohol to doubly activated 21 to give 28 (seeFIG. 15). Compound 21 (330 mg, 0.612 mmol) was dissolved in DMF (5 mL)and p-aminobenzyl alcohol (166 mg, 1.35 mmol), DIPEA (214 μL, 1.22mmol), and 1-hydroxybenzotriazole (24.8 mg, 0.184 mmol) were added atroom temperature. The reaction mixture was stirred at room temperaturefor 2 days. The reaction mixture was concentrated under reduced pressureand then dissolved in ethyl acetate (75 mL). The clear solution waswashed with a 10% aqueous citric acid solution and brine, dried withanhydrous sodium sulfate, filtered, and concentrated under reducedpressure. Column chromatography (chloroform/MeOH 8/1) afforded 28 (181mg, 0.357 mmol, 58%) as a white solid after freeze-drying. ¹H-NMR (300MHz, CD₃OD/CDCl₃) δ 4.57 (s, 4H, CH₂OH), 4.89 (s, 2H, CH₂OC(O)R), 4.92(s, 2H, CH₂OC(O)R), 6.95 (s, 1H, CH═CR₂), 7.25-7.54 (m, 10H, aromatic),8.24 (d, 2H, J=8.8 Hz, aromatic) ppm; ESI-MS m/e 530 (M+Na)⁺; Anal.(C₂₆H₂₅N₃O₈) calculated C 61.53%, H 4.97%, N 8.28%, measured C 61.60%, H5.20%, N 7.61%.

Example 15

Activation of diol 28 with p-nitrophenyl chloroformate to give 29 (seeFIG. 15). To a solution of compound 28 (160 mg, 0.315 mmol) in THF (5mL) were added DIPEA (440 μL, 2.52 mmol), p-nitrophenyl chloroformate(381 mg, 1.89 mmol), and pyridine (12.9 μL, 0.158 mmol) at 0° C. Thereaction mixture was slowly warmed to room temperature and stirredovernight. Ethyl acetate (75 mL) was added to the reaction mixture andthe mixture was washed with a saturated aqueous sodium bicarbonatesolution, a 10% aqueous citric acid solution, and brine. The organicfraction was dried with anhydrous sodium sulfate, filtered, andconcentrated under reduced pressure. Column chromatography(EtOAc/hexanes 1/1) afforded 29 (155 mg, 0.187 mmol, 59%) as a whitesolid after freeze-drying. ¹H-NMR (300 MHz, CDCl₃/DMSO-D₆) δ 4.90 (s,2H, CH₂OC(O)NHR), 4.95 (s, 2H, CH₂OC(O)NHR), 5.23 (s, 4H, CH₂OC(O)OR),6.96 (s, 1H, CH═CR₂), 7.34-7.56 (m, 14H, aromatic), 8.22-8.28 (m, 6H,aromatic) ppm; ESI-MS m/e 860 (M+Na)⁺, 1697 (2M+Na)⁺; Anal.(C₄₀H₃₁N₅O₁₆·1.5H₂O) calculated C 55.56%, H 3.96%, N 8.10%, measured C55.60%, H 3.96%, N 8.30%.

Example 16

Coupling of p-methoxybenzylamine to doubly activated 29 to give 30 (seeFIG. 16). To a solution of 29 (50 mg, 59.7 μmol) in1-methyl-2-pyrrolidinone (4 mL) were added at 0° C. p-methoxybenzylamine(31 μL, 0.24 mmol) and DIPEA (3.0 μL, 18 μmol). The reaction mixture wasstirred at room temperature for 15 h. A 10% solution of isopropanol inethyl acetate (25 mL) was added and the resulting mixture was washedwith water and brine, dried with anhydrous sodium sulfate, filtered, andconcentrated under reduced pressure. Column chromatography(EtOAc/hexanes 1/3) gave 30 (35.0 mg, 42.0 μmol, 70%) as a white solid.¹H-NMR (300 MHz, CDCl₃/CD₃OD) δ 3.79 (s, 6H, OCH₃), 4.26 (s, 4H, NCH₂),4.90 (s, 2H, CH₂OC(O)NHC₆H₄R), 4.94 (s, 2H, CH₂OC(O)NHC₆H₄R), 5.04 (s,4H, CH₂OC(O)NCH₂R), 6.85 (d, 4H, J=8.6 Hz, aromatic), 6.96 (s, 1H,CH═CR₂), 7.19-7.28 (m, 8H, aromatic), 7.37-7.42 (m, 4H, aromatic), 7.53(d, 2H, J=8.6 Hz, aromatic), 8.25 (m, 2H, aromatic) ppm; FAB-MS m/e 856[M+Na]⁺, 1690 [2M+Na]⁺.

Example 17

Coupling of p-chlorobenzylamine to doubly activated 29 to give 31 (seeFIG. 16). To a solution of 29 (72 mg, 86 μmol) in THF (4 mL) were addedat 0° C. p-chliorobenzylaniine (42 μL, 0.34 mmol) and DIPEA (4.5 μL, 26μmol). The reaction mixture was stirred at room temperature for 15 h.The reaction mixture was concentrated under reduced pressure and theresidue was purified by colum chromatography (EtOAc/hexanes 1/1) toafford 31 (60 mg, 71.2 mmol, 83%) as a white solid after freeze-drying.¹H-NMR (300 MHz, CDCl₃/CD₃OD) δ 4.27 (s, 4H, NCH₂), 4.89 (s, 2H,CH₂OC(O)NHC₆H₄R), 4.94 (s, 2H, CH₂OC(O)NHC₆H₄R), 5.03 (s, 4H,CH₂OC(O)NCH₂R), 6.98 (s, 1H, CH═CR₂), 7.21-7.29 (m, 12H, aromatic),7.40-7.44 (m, 4H, aromatic), 7.57 (d, 2H, J=8.6 Hz, aromatic), 8.25 (m,2H, aromatic) ppm; FAB-MS m/e 864 [M+Na]⁺.

Example 18

Coupling of phenethyl alcohol to doubly activated 29 to give 32 (seeFIG. 16). To a solution of 29 (18 mg, 21 μmol) in THF (2 mL) were addedat 0° C. phenethyl alcohol (10.3 μL, 85.9 μmol), DIPEA (1.9 μL, 11μmol), and DMAP (10.5 mg, 85.9 μmol). The reaction mixture was stirredat room temperature for 15 h. The reaction mixture was concentratedunder reduced pressure and the residue was purified by columnchromatography (EtOAc/hexanes 1/1) to afford 32 (9.5 mg, 12 mmol, 55%)as a white solid after freeze-drying. ¹H-NMR (300 MHz, CDCl₃/CD₃OD) δ2.97 (t, 4H, J=7.0 Hz, CH₂CH₂Ph), 4.34 (t, 4H, J=7.0 Hz, CH₂CH₂Ph), 4.90(s, 2H, CH₂OC(O)NHC₆H₄R), 4.94 (s, 2H, CH₂OC(O)NHC₆H₄R), 5.07 (s, 4H,CH₂OC(O)OR), 6.97 (s, 1H, CH═CR₂), 7.19-7.30 (m, 14H, aromatic),7.39-7.44 (m, 4H, aromatic), 7.54 (d, 2H, J=8.7 Hz, aromatic), 8.25 (d,2H, J=8.7 Hz, aromatic) ppm; FAB-MS m/e 804 [M+H]⁺, 826 [M+Na]⁺.

Example 19

Coupling of benzylamine to 4-nitrobenzyl chloroformate 33 to give 34(see FIG. 17). A solution of chloroformate 33 (200 mg, 0.923 mmol) inTHF was added dropwise to a mixture of benzylamine (101 μL, 0.923 mmol)and Et3N (129 μL, 1 equiv) in THF at 0° C. The reaction mixture wasallowed to reach room temperature and was stirred for 16 h. EtOAc wasadded and the organic layer was washed with 0.1 M NaOH, 10% citric acid,and brine, and dried over sodium sulfate to quantitatively yield desiredproduct 34. ¹H-NMR (300 MHz, CDCl₃) δ 4.39 (d, 2H, J=6.0 Hz, benzylicbenzylamine), 5.22 (s, 2H, benzylic spacer), 7.25-7.40 (m, 5H, aromaticbenzylamine), 7.50 (d, 2H, J=8.4 Hz, aromatic spacer), 8.20 (d, 2H,J=8.4 Hz, aromatic spacer) ppm.

Example 20

Proof of principle for triple elimination. Formation of benzyl alcoholupon reduction of 13 (see FIG. 19). Compound 13 was dissolved in 3 mL ofMeOH/AcOH/THF (1.5/0.5/1). Zinc powder was added and the reactionmixture was stirred at Rt. After 30 min thin layer chromatographyindicated disappearance of starting compound and formation of asubstantial amount of benzyl alcohol. The mixture was filtered overhyflo and the residue was washed with MeOH/dichloromethane. The filtratewas concentrated in vacuo, dioxane was added, and the resulting solutionwas freeze-dried. ¹H-NMR (300 MHz, CDCl₃/CD₃OD/CD₃CN/DMSO-D₆) δ 4.57 (s,2H, benzylic), 7.28-7.36 (m, 5H, aromatic) ppm, identical with peaksobserved for free benzyl alcohol in the same medium.

Example 21

Proof of principle for triple elimination. Formation of phenethylalcohol upon reduction of 14 (see FIG. 19). Compound 14 was dissolved in3.75 mL of MeOH/AcOH/THF (2/0.75/1). Zinc powder was added and thereaction mixture was stirred at Rt. After 1 h thin layer chromatographyindicated disappearance of starting compound and formation of asubstantial amount of phenethyl alcohol. The mixture was filtered overhyflo and the residue was washed with MeOH/dichloromethane. The filtratewas concentrated in vacuo, dioxane was added, and the resulting solutionwas freeze-dried. ¹H-NMR (300 MHz, DMSO-D₆/CD₃OD/D₂O) δ 2.68 (t, 2H,CH₂CH ₂Ph), 3.58 (t, 2H, CH ₂CH₂Ph), 7.11-7.22 (m, 5H, aromatic) ppm,identical with peaks observed for free phenethyl alcohol in the samemedium; ESI-MS m/e 122 (M)⁺.

Example 22

Reduction of the nitro group in 15. Failure of elimination ofbenzylamine (see FIG. 20). Compound 15 (16 mg) was dissolved in 10 mL ofMeOH/AcOH (8/2). Zinc powder was added and the reaction mixture wasstirred at Rt. After 30 min, thin layer chromatography indicateddisappearance of starting compound and showed complete conversion to asingle product. Formation of benzylamine was not or was hardly observed.The mixture was filtered over hyflo and the residue was washed withMeOH/dichloromethane. The filtrate was concentrated in vacuo, themixture was freeze-dried. Under a number of varied circumstances(different deuterated solvent combinations, different pH (acidic,neutral, and basic), different buffers), ¹H-NMR did hardly showformation of free benzylamine. After 2 days, maximally 33 percent offree benzylamine was observed (¹H-NMR). Dissolving the reduced productin DMF did not increase amounts of released benzylamine. Reduction of 15using Raney Nickel/hydrazine did not lead to different results.

Example 23

Deprotection of the Aloc group from 19. Failure of elimination ofpropylamine (see FIG. 21). Compound 19 (36 mg, 69 μmol) was dissolved in1 mL of CDCl₃. Morpholine was added (20 equiv, 120 μL, 1.38 mmol), andargon was bubbled through the solution. Then, a catalytic amount ofpalladium tetrakistriphenylphosphine was added. Elimination ofpropylamine was studied by ¹H-NMR. After 1 h, thin layer chromatographyindicated disappearance of starting compound. Under a number of variedcircumstances (different deuterated solvent combinations, different pH(acidic, neutral, and basic), different buffers, in the presence ofdifferent nucleophiles), by ¹H-NMR it was shown that maximally 33percent of free propylamine had been formed after 2 weeks.

Example 24

Proof of principle for double elimination. Formation of paclitaxel uponreduction of 22 (see FIG. 22). Nitrocinnamyl biscarbonate 22 wasdissolved in 3.5 mL of MeOH/AcOH/THF (2/0.5/1). Zinc powder was addedand the reaction mixture was stirred at Rt. After 30 min thin layerchromatography indicated complete disappearance of starting compound andformation of a substantial amount of paclitaxel. The mixture wasfiltered over hyflo and the residue was washed withMeOH/dichloromethane. The filtrate was concentrated in vacuo, dioxanewas added and the resulting solution was freeze-dried. Distinguishingpeaks in the ¹H-NMR, (300 MHz, CD₃OD) δ 4.19 (s, 4H, 20a and 20b), 4.32(dd, 2H, 7), 4.74 (d, 2H, 2′), 4.99 (dd, 2H, 5), 5.65 (m, 4H, 2 and 3′),6.16 (t, 2H, 13), 6.45 (s, 2H, 10) ppm, were identical with peaksobserved for free paclitaxel in the same medium. The proton NMR spectrumdid not show 2′-coupled paclitaxel; only unconjugated paclitaxel waspresent; FAB-MS m/e 854 (M+H)⁺, 876 (M+Na)⁺.

Example 25

Proof of principle for quadruple elimination. Formation of paclitaxelupon reduction of 26 (see FIG. 23). Compound 26 was dissolved in 3.25 mLMeOH/AcOH/TF (1.5/0.75/1). Zinc powder was added and the reactionmixture was stirred. After 30 min, thin layer chromatography indicateddisappearance of starting compound and formation of a substantial amountof paclitaxel. After 16 h, the mixture was filtered over hyflo and theresidue was washed with MeOH/dichloromethane. The filtrate wasconcentrated in vacuo, dioxane was added, and the resulting solution wasfreeze-dried. Distinguishing peaks in the ¹H-NMR, (300 MHz,CD₃OD/CDCl₃/DMSO-D₆) δ 4.77 (d, 4H, 2′), 4.98 (d, 4H, 5), 5.65 (m, 8H, 2and 3′), 6.17 (m, 4H, 13), 6.39 (s, 4H, 10) ppm, were identical withpeaks observed for free paclitaxel in the same medium; ESI-MS m/e 877(M+Na)⁺.

Example 26

Reduction of the nitro group in 27. Failure of elimination ofbenzylamine (see FIG. 24). To a solution of 27 (18 mg, 38 μmol) in a 4:1mixture of MeOH and AcOH (7.5 mL) Zinc powder was added and the reactionmixture was stirred at Rt. After 1 h, thin layer chromatographyindicated disappearance of starting compound and showed completeconversion to a single product. No formation of benzylamine wasobserved. The mixture was filtered over hyflo and the residue was washedwith MeOH/dichloromethane. The filtrate was concentrated in vacuo,dioxane was added and the mixture was freeze-dried. In a number ofdifferent deuterated solvent combinations, possessing a different pH, insome cases buffered, in the presence or absence of nucleophiles, ¹H-NMRdid not show formation of more than 50 percent of free benzylamine, evenafter a day.

Example 27

Reduction of the nitro group in 30. Failure of elimination ofp-methoxybenzylamine (see FIG. 25). To a solution of 30 (10 mg, 12 μmol)in a 1:1 mixture of THF and methanol (2 mL) were added Raney Nickel (50%slurry in water, 0.05 mL) and hydrazine monohydrate (1.0 μL, 21 μmol).Thin layer chromatography (EtOAc/heptanes 3/1) showed completeconversion to a single product within 20 minutes. The reaction mixturewas stirred overnight at room temperature. No formation ofp-methoxybenzylamine was observed after night. The reaction mixture wasfiltered and concentrated under reduced pressure. The residue wasdissolved in a 1:1 mixture of DMSO-d₆ and D₂O. ¹H-NMR did not show anyformation of free p-methoxybenzylamine after 1, 5, or 20 days. Slowdecomposition of reduced 30 not involving release ofp-methoxybenzylamine was observed.

Example 28

Reduction of the nitro group in 31. Failure of elimination ofp-chlorobenzylamine (see FIG. 25). To a solution of 31 (10 mg, 12 μmol)in a 1:1 mixture of THF and methanol (2 mL) were added Raney Nickel (50%slurry in water, 0.05 mL) and hydrazine monohydrate (0.6 μL, 12 μmol).Thin layer cohromatography (EtOAc/heptanes 3/1) showed completeconversion to a single product within 30 minutes. The reaction mixturewas filtered and concentrated under reduced pressure. The residue wasdissolved in a 1:1 mixture of CDCl₃ and CD₃OD. ¹H-NMR did not show anyformation of free p-methoxybenzylamnine after 2 h, 15 h, 2 days, or 5days. Slow decomposition of reduced 31 not involving release ofp-chlorobenzylamine was observed.

Example 29

Reduction of the nitro group in 32. Formation of phenethyl alcohol (seeFIG. 26). To a solution of 32 (8.0 mg, 10 μmol) in a 1:1 mixture of THFand methanol (1.5 mL) were added Raney Nickel (50% slurry in water, 0.05mL) and hydrazine monohydrate (0.5 μL, 10 μmol). After 20 minutes,hydrazine monohydrate (0.5 μL, 10 μmol) were added. Thin layerchromatography (EtOAc/heptanes 1/1) showed complete conversion to asingle product after 1 h. The reaction mixture was filtered andconcentrated under reduced pressure. The residue was dissolved in a 1:1mixture of CDCl₃ and CD₃OD to which 10 drops of DMSO-d₆ were added.¹H-NMR showed complete release of free phenethyl alcohol. Distinguishingpeaks in the ¹H-NMR spectrum, (300 MHz, CD₃OD/CDCl₃/DMSO-D₆) δ 2.83 (t,2H, J=7.1 Hz, CH₂CH₂Ph) and 3.76 (t, 2H, J=7.1 Hz, CH₂CH₂Ph) ppm, wereidentical with peaks observed for phenethyl alcohol in the same medium.

Example 30

Proof of principle for single elimination. Formation of benzylamine uponreduction of 34 (see FIG. 27). Compound 34 (40 mg) was dissolved in 3.75mL of MeOH/AcOH/THF (2/0.75/1). Zinc powder was added and the reactionmixture was stirred at Rt. After 100 min, thin layer chromatographyindicated disappearance of starting compound and formation of asubstantial amount of benzylamine. The mixture was filtered over hyfloand the residue was washed with MeOH/dichloromethane. The filtrate wasconcentrated in vacuo. ¹H-NMR (300 MHz, CD₃OD/CDCl₃/DMSO-D₆) δ 3.98 (s,2H, benzylic, liberated benzylamine), 7.34-7.43 (m, 5H, aromatic,liberated benzylamine) ppm, identical with peaks observed for freebenzylamine in the same medium, indicated complete release of freebenzylamine. NMR signals from conjugated benzylamine have disappeared.

Example 31

Stability of 35. Treatment of 35 with Zinc (see FIG. 28). Dibenzylcarbonate 35 (13 mg) was dissolved in 2.5 mL MeOH/THF/AcOH(1.5/0.5/0.5). Zinc powder was added and the reaction mixture wasstirred at Rt for 16 h. Thin layer chromatography indicated no formationof new compounds, and indicated stability of 35. The mixture wasfiltered over hyflo and the residue was washed withMeOH/dichloromethane. The filtrate was concentrated in vacuo. ¹H-NMR(300 MHz, CDCl₃/CD₃OD/DMSO-D₆) δ 5.07 (s, 4H, benzylic), 7.27 (m, 10H,aromatic) ppm, showed that the product was identical to startingcompound; no benzyl alcohol was observed.

Example 32

Stability of 36. Treatment of 36 with Zinc (see FIG. 28).Paclitaxel-2′-carbonate 36 (3 mg) was dissolved in 2.5 mL MeOH/THF/AcOH(1.5/0.5/0.5). Zinc powder was added and the reaction mixture wasstirred at Rt for 16 h. Thin layer chromatography indicated no formationof new compounds, and indicated stability of 36. The mixture wasfiltered over hyflo and the residue was washed withMeOH/dichloromethane. The filtrate was concentrated in vacuo.Distinguishing peaks in the ¹H-NMR spectrum, (300 MHz,CDCl₃/CD₃OD/DMSO-D₆) δ 4.81 (d, 2H, J=7.2 Hz, CH ₂ cinnamyl), 5.38 (d,1H J=8.7 Hz, 2′) ppm, showed that the product was identical to startingcompound; no cinnamyl alcohol or free paclitaxel was observed.

Example 33

Cytoxicity of multiple release spacers 16 and 23. The anti-proliferativeeffect of the monomeric spacers was determined in vitro applying sevenwell-characterised human tumor cell lines and the microculturesulphorhodamine B (SRB) test. The anti-proliferative effects weredetermined and expressed as ID₅₀ values (ng/mL), which are the (pro)drugconcentrations that gave 50% inhibition of cell growth when compared tocontrol cell growth after 5 days of incubation. Triple release monomer16: cell lines (ID₅₀ values in ng/mL) MCF-7; breast cancer (>62.500).EVSA-T; breast cancer (>62.500). WIDR; colon cancer (>62.500). IGROV;ovarian cancer (>62.500). M19; melanoma (>62.500). A498; renal cancer(55867). H226; non-small cell lung cancer (>62.500). Double releasemonomer 23: cell lines (ID₅₀ values in ng/mL): MCF-7; breast cancer(>62.500). EVSA-T; breast cancer (>62.500). WIDR; colon cancer(>62.500). IGROV; ovarian cancer (>62.500). M19; melanoma (>62.500).A498; renal cancer (56.784). H226; non-small cell lung cancer (42.406).

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35 Remington's Pharmaceutical Science (15th ed., Mack Publishing,Easton, Pa., 1980) (incorporated by reference in its entirety for allpurposes)

1. A compound comprising a specifier (V) linked to two or more of thesame or different leaving groups (Z) via a self-eliminating multiplerelease spacer or spacer system, which compound upon a single activationstep releases at least two leaving groups, said activation step beingthe removal or transformation of the specifier.
 2. A compound accordingto claim 1 comprising two or more self-eliminating multiple releasespacers.
 3. A compound according to claim 1 or 2 comprising aself-eliminating multiple release spacer system incorporating two ormore generations of self-eliminating multiple release spacers in theform of a dendritic structure.
 4. A compound according to claim 1, 2 or3 having a formula selected fromV—(W—)_(w)(X—)_(x)C((A—)_(a)Z)_(c),V—(W—)_(w)(X—)_(x)C(D((A—)_(a)Z)_(d))_(c),V—(W—)_(w)(X—)_(x)C(D(E((A—)_(a)Z)_(e))_(d))_(c), andV—(W—)_(w)(X—)_(x)C(D(E(F((A—)_(a)Z)_(f))_(e))_(d))_(c), wherein: V isselected from [O] and a specifier which is removed or transformed by achemical, photochemical, physical, biological, or enzymatic activation,optionally after prior binding to a receptor;(W—)_(w)(X—)_(x)C((A—)_(a))_(c),(W—)_(w)(X—)_(x)C(D((A—)_(a))_(d))_(c),(W—)_(w)(X—)_(x)C(D(E((A—)_(a))_(e))_(d))_(c), and(W—)_(w)(X—)_(x)C(D(E(F((A—)_(a))_(f))_(e))_(d))_(c) areself-eliminating multiple release spacers or spacer systems; W and X areeach a single release 1,(4+2n) electronic cascade spacer, being the sameor different; A is a cyclization elimination spacer; C, D, E, and F areeach a self-eliminating multiple release spacer or spacer system thatupon activation can maximally release c, d, e, and f leaving groups,respectively; each Z is independently a leaving group or H or OH or areactive moiety; a is 0 or 1; c, d, e, and f are independently aninteger from 2 (included) to 24 (included); w and x are independently aninteger from 0 (included) to 5 (included); n is an integer of 0(included) to 10 (included).
 5. A compound according to any of thepreceding claims, wherein the self-elimination multiple release spacersor spacer systems C, D, E, and F are independently selected fromcompounds having the formula

Wherein B is selected from NR¹, O, and S; P isC(R²)(R³)Q—(W—)_(w)(X—)_(x); wherein Q has no meaning or is —O—CO—; Wand X are each a single release 1,(4+2n) electronic cascade spacer,being the same or different; G, H, I, J, K, L, M, N, and O areindependently selected from compounds having the formula:

wherein R¹, R², R³, R⁴, and R⁵ independently represent H, C₁₋₆ alkyl,C₃₋₂₀ heterocyclyl, C₅₋₂₀ aryl, C₁₋₆ alkoxy, hydroxy (OH), amino (NH₂),mono-substituted amino (NR_(x)H), di-substituted amino (NR_(x) ¹R_(x)²), nitro (NO₂), halogen, CF₃, CN, CONH₂, SO₂Me, CONHMe, cyclic C₁₋₅alkylamino, imidazolyl, C₁₋₆ alkylpiperazinyl, morpholino, thiol (SH),thioether (SR_(x)), tetrazole, carboxy (COOH), carboxylate (COOR_(x)),sulphoxy (S(═O)₂OH), sulphonate (S(═O)₂OR_(x)), sulphonyl (S(═O)₂R_(x)),sulphixy (S(═O)OH), sulphinate (S(═O)OR_(x)), sulphinyl (S(═O)R_(x)),phosphonooxy (OP(═O)(OH)₂), and phosphate (OP(═O)(OR_(x))₂), whereR_(x), R_(x) ¹ and R_(x) ² are independently selected from a C₁₋₆ alkylgroup, a C₃₋₂₀ heterocyclyl group or a C₅₋₂₀ aryl group, two or more ofthe substituents R¹, R², R³, R⁴, and R⁵ optionally being connected toone another to form one or more aliphatic or aromatic cyclic structures,or G, J, and M may also be selected from the group of P and hydrogenwith the proviso that if two of G, J, and M are hydrogen, the remaininggroup must be

or be

and at the same time be conjugated to

g, h, i, j, k, l, m, n, o, h′, g′, k′, j′, n′, m′ are independently 0,1, or 2 with the provisos that if G=hydrogen or P, g, h, i, h′, and g′all equal 0; if J=hydrogen or P, j, k, l, k′, and j′ all equal 0; ifM=hydrogen or P, m, n, o, n′, and m′ all equal 0; if G, H, I, J, K, L,M, N, or O is

then g+g′=1, h+h′=1,i=1, j+j′=1, k+k′=1, l=1,m+m′=1,n+n′=1, or o=1,respectively; if G, H, I, J, K, L, M, N, or O is

then g+g′=2, h+h′=2, i=2,j+j′=2, k+k′=2, l=2, m+m′=2, n+n′=2, or o=1,respectively; if g′=0 and G is not hydrogen or P, then h, h′, and iequal 0 and g>0; if g=0 and G is not hydrogen or P, then g′>0; if g′>0and h′=0, then i=0 and h>0; if g′>0 and h=0, then h′>0 and i>0; if j′=0and J is not hydrogen or P, then k, k′, and 1 equal 0 and j>0; if j=0and J is not hydrogen or P, then j′>0; if j′>0 and k′=0, then l=0 andk>0; if j′>0 and k=0,then k′>0 and l>0; if m′=0 and M is not hydrogen orP, then n, n′, and o equal 0 and m>0; if m=0 and M is not hydrogen or P,then m′>0; if m′>0 and n′=0, then o=0 and n>0; if m′>0 and n=0,then n′>0and o>0; w and x are independently an integer from 0 (included) to 5(included); with the proviso that if the compound contains only C and noD, no E, and no F are present, and B=NR¹, and G and M are H, and g, h,i, h′, g′, k, l, k′, l′, m, n, o, n′, and m′ are 0, and J=

and j=2, and Q=—O—CO—, and w and x are 0, and R¹, R², R³, and R⁴ are H,then at least one of the leaving groups Z is not connected to Q via analiphatic amino group.
 6. A compound according to any of the precedingclaims, wherein the 1,(4+2n) electronic cascade spacers W and X areindependently selected from compounds having the formula

Q′=—R⁵C═CR⁶—, S, O, NR⁵, —R⁵C═N—, or —N═CR⁵—B=NR⁷, O, SP=C(R³)(R⁴)Q wherein Q has no meaning or is —O—CO—; t, u, and y areindependently an integer of 0 to 5; T, U, and Y are independentlyselected from compounds having the formula:

wherein R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, and R⁹ independently representH, C₁₋₆ alkyl, C₃₋₂₀ heterocyclyl, C₅₋₂₀ aryl, C₁₋₆ alkoxy, hydroxy(OH), amino (NH₂), mono-substituted amino (NR_(x)H) di-substituted amino(NR_(x) ¹R_(x) ²), nitro (NO₂), halogen, CF₃, CN, CONH₂, SO₂Me, CONHMe,cyclic C₁₋₅ alkylamino, imidazolyl, C₁₋₆ alkylpiperazinyl, morpholino,thiol (SH), thioether (SR_(x)), tetrazole, carboxy (COOH), carboxylate(COOR_(x)), sulphoxy (S(═O)₂OH), sulphonate (S(═O)₂OR_(x)), sulphonyl(S(═O)₂R_(x)), sulphixy (S(═O)OH), sulphinate (S(═O)OR_(x)), sulphinyl(S(═O)R_(x)), phosphonooxy (OP(═O)(OH)₂), and phosphate(OP(═O)(OR_(x))₂), where R_(x), R_(x) ¹ and R_(x) ² are independentlyselected from a C₁₋₆ alkyl group, a C₃₋₂₀ heterocyclyl group or a C₅₋₂₀aryl group, two or more of the substituents R¹, R², R³, R⁴, R⁵, R⁶, R⁷,R⁸, or R⁹ optionally being connected to one another to form one or morealiphatic or aromatic cyclic structures.
 7. A compound according to anyof the preceding claims wherein the leaving groups Z are linked to theself-eliminating multiple release spacer or spacer system via an O, S,or aromatic N of the leaving group.
 8. A compound according to any ofthe preceding claims wherein the leaving groups Z are linked to theself-eliminating multiple release spacer or spacer system via analiphatic N and wherein at least one multiple release spacer or spacersystem of either generation C, D (if present), E (if present), or F (ifpresent) is a phenol- or thiophenol-based multiple release spacer orspacer system, meaning that i) B=O or S for at least one multiplerelease spacer in said generation, or ii) when B=N for all multiplerelease spacers in said generation, at least one single release spaceris connected to at least two branches of at least one multiple releasespacer in said generation, and B=O or S for at least two of said singlerelease spacers.
 9. A compound according to claim 8 wherein B=O or S forall multiple release spacers or spacer systems in said generation.
 10. Acompound according to claims 8 or 9, wherein the phenol- orthiophenol-based multiple release spacers are connected to either A or Zor S, wherein S is as defined in claim
 26. 11. A compound according toany of the preceding claims, wherein the ω-amino aminocarbonylcyclization elimination spacer A is a compound having the formula:

wherein: a is an integer of 0 or 1; and b is an integer of 0 or 1; and cis an integer of 0 or 1; provided that a+b+c=2 or3; and wherein R¹ andR² independently represent H, C₁₋₆ alkyl, said alkyl being optionallysubstituted with one or more of the following groups: hydroxy (OH),ether (OR_(x)), amino (NH₂), mono-substituted amino (NR_(x)H),di-substituted amino (NR_(x) ¹R_(x) ²), nitro (NO₂), halogen, CF₃, CN,CONH₂, SO₂Me, CONHMe, cyclic C₁₋₅ alkylamino, imidazolyl, C₁₋₆alkylpiperazinyl, morpholino, thiol (SH), thioether (SR_(x)), tetrazole,carboxy (COOH), carboxylate (COORs), sulphoxy (S(═O)₂OH), sulphonate(S(═O)₂OR_(x)), sulphonyl (S(═O)₂R_(x)), sulphixy (S(═O)OH), sulphinate(S(═O)OR_(x)), sulphinyl (S(═O)R_(x)), phosphonooxy (OP(═O)(OH)₂), andphosphate (OP(═O)(OR_(x))₂), where R_(x), R_(x) ¹ and R_(x) ² areselected from a C₁₋₆ alkyl group, a C₃₋₂₀ heterocyclyl group or a C₅₋₂₀aryl group; and R³, R⁴, R⁵, R⁶, R⁷, and R⁸ independently represent H,C₁₋₆ alkyl, C₃₋₂₀ heterocyclyl, C₅₋₂₀ aryl, C₁₋₆ allkoxy, hydroxy (OH),amino (NH₂), mono-substituted amino (NR_(x)H), di-substituted amino(NR_(x) ¹R_(x) ²), nitro (NO₂), halogen, CF₃, CN, CONH₂, SO₂Me, CONHMe,cyclic C₁₋₅ alkylamino, imidazolyl, C₁₋₆ alkylpiperazinyl, morpholino,thiol (SH), thioether (SR_(x)), tetrazole, carboxy (COOH), carboxylate(COOR_(x)), sulphoxy (S(═O)₂OH), sulphonate (S(═O)₂OR_(x)), sulphonyl(S(═O )₂R_(x)), sulphixy (S(═O)OH), sulphinate (S(═O)OR_(x)), sulphinyl(S(═O)R_(x)), phosphonooxy (OP(═O)(OH)₂), and phosphate(OP(═O)(OR_(x))₂), where R_(x), R_(x) ² and R 2 are selected from a C₁₋₆alkyl group, a C₃₋₂₀ heterocyclyl group or a C₅₋₂₀ aryl group; andwherein R¹, R², R³, R⁴, R⁵, R⁶, R⁷, and R⁸ can be a part of one or morealiphatic or aromatic cyclic structures, two or more of the substituentsR¹, R², R³, R⁴, R⁵, R⁶, R⁷, or R⁸ optionally being connected to oneanother to form one or more aliphatic or aromatic cyclic structures. 12.A compound according to any of the preceding claims, wherein group A isan ω-amino aminocarbonyl cyclization spacer, and Z is a moiety coupledvia its hydroxyl group to A.
 13. A compound according to any of thepreceding claims wherein w+x>0.
 14. A compound according to any of thepreceding claims wherein(W—)_(w)(X—)_(x)C_(c),(W—)_(w)(X—)_(x)C(D_(d))_(c),(W—)_(w)(X—)_(x)C(D(E_(e))_(d))_(c) or(W—)_(w)(X—)_(x)C(D(E(F_(f))_(e))_(d))_(c) is selected from the groupconsisting of

and from the compounds depicted above wherein single release1,6-elimination p-aminobenzyloxycarbonyl spacer(s) are replaced bysingle release 1,8-elimination p-aminocinnamyloxycarbonyl spacer(s). 15.A compound according to claim 14 which further comprises ω-aminoaminocarbonyl cyclization spacers A.
 16. A compound according to any ofthe preceding claims wherein the specifier V contains a substrate thatcan be cleaved by plasmin, one of the cathepsins, cathepsin B,β-glucuronidase, prostate-specific antigen (PSA), urokinase-typeplasminogen activator (u-PA), a member of the family of matrixmetalloproteinases, or wherein the specifier V is [O] or contains anitro-(hetero)aromatic moiety that can be removed or transformed byreduction under hypoxic conditions or by reduction by a nitroreductase.17. A compound according to any of the preceding claims wherein Z isselected from an antibiotic, an anti-inflammatory agent, an anti-viralagent, and preferably an anticancer agent.
 18. The compound of claim 17wherein Z is selected from (hydroxyl containing cytotoxic compounds)etoposide, combrestatin, camptothecin, irinotecan (CPT-11), SN-38,topotecan, 9-aminocamptothecin, 9-nitrocamptothecin,10-hydroxycamptothecin, GG211, lurtotecan, paclitaxel, docetaxel,esperamycin, 1,8-dihydroxy-bicyclo[7.3.1]trideca-4-ene-2,6-diyne-13-one,anguidine, doxorubicin, morpholine-doxorubicin, N-(5,5-diacetoxypentyl)doxorubicin, daunorubicin, epirubicin, idarubicin, mitoxantrone,vincristine, vinblastine, tallysomycin, bleomycin,4-bis(2-chloroethyl)aminophenol, 4-bis(2-fluoroethyl)aminophenol, andderivatives thereof, (sulfhydryl containing compounds) esperamicin and6-mercaptopurine, and derivatives thereof, (carboxyl containingcompounds) methotrexate, aminopterin, camptothecin (ring-opened form ofthe lactone), chlorambucil, melphalan, butyric acid and retinoic acid,and derivatives thereof, and (aziridine amino containing or aromaticamino containing compounds) mitomycin C, mitomycin A, an anthracyclinederivative containing an amine functionality with sufficient leavinggroup ability, mitoxantrone, 9-amino camptothecin, methotrexate,aminopterin, tallysomycin, bleomycin, actinomycin,N,N-bis(2-chloroethyl)-p-phenylenediamine,N,N-bis(2-fluoroethyl)-p-phenylenediamine, deoxycytidine, cytosinearabinoside, gemcitabine, and derivatives thereof, and (aliphatic aminocontaining compounds) daunorubicin, doxorubicin, epirubicin, idarubicin,N-(5,5-diacetoxypentyl)doxorubicin, an anthracycline, N⁸-acetylspermidine, 1-(2-chloroethyl)-1,2-dimethanesulfonyl hydrazine, orderivatives thereof.
 19. A compound according to claim 18 wherein Zrepresents paclitaxel, docetaxel, or a derivative thereof, which iscoupled to the self-eliminating multiple release spacer or spacer systemvia its 2′-hydroxyl group.
 20. A compound according to claim 18 whereinZ represents camptothecin, irinotecan (CPT-11), SN-38, topotecan,9-aminocamptothecin, 9-nitrocamptothecin, 10-hydroxycamptothecin, GG211, lurtotecan, or a derivative thereof, which is coupled to theself-eliminating multiple release spacer or spacer system via its20-hydroxyl group.
 21. A compound according to claim 18 wherein Zrepresents SN-38, topotecan, 10-hydroxycamptothecin, etoposide,4-bis(2-chloroethyl)aminophenol, 4-bis(2-fluoroethyl)aminophenol, or aderivative thereof, which is coupled to the self-eliminating multiplerelease spacer or spacer system via its phenolic hydroxyl group.
 22. Acompound according to claim 18 wherein Z represents 9-aminocamptothecin,N,N-bis(2-chloroethyl)-p-phenylenediamine,N,N-bis(2-fluoroethyl)-p-phenylenediamine, or a derivative thereof,which is coupled to the self-eliminating multiple release spacer orspacer system via its aromatic primary amine group.
 23. A compoundaccording to claim 18 wherein Z represents daunorubicin, doxorubicin,epirubicin, idarubicin, N-(5,5-diacetoxypentyl)doxorubicin, ananthracycline, N⁸-acetyl spermidine,1-(2-chloroethyl)-1,2-dimethanesulfonyl hydrazine, or derivativesthereof, which is coupled to the self-eliminating multiple releasespacer or spacer system via its primary aliphatic amino group andwherein at least one multiple release spacer or spacer system of eithergeneration C, D (if present), E (if present), or F (if present) is aphenol- or thiophenol-based multiple release spacer or spacer system,meaning that i) B=O or S for at least one multiple release spacer insaid generation, or ii) when B=N for all multiple release spacers insaid generation, at least one single release spacer is connected to atleast two branches of at least one multiple release spacer in saidgeneration, and B=O or S for at least two of said single releasespacers.
 24. A compound according to claim 23 wherein B=O or S for allmultiple release spacers or spacer systems in said generation.
 25. Acompound according to claims 23 or 24, wherein the phenol- orthiophenol-based multiple release spacers are connected to either A or Zor S, wherein S is as defined in claim
 26. 26. A compound having aformula selected fromV—(W—)_(w)(X—)_(x)C((A—)_(a)S)_(c),V—(W—)_(w)(X—)_(x)C(D((A—)_(a)S)_(d))_(c),V—(W—)_(w)(X—)_(x)C(D(E((A—)_(a)S)_(e))_(d))_(c), andV—(W—)_(w)(X—)_(x)C(D(E(F((A—)_(a)S)_(f))_(e))_(d))_(c), wherein: V, W,X, C, D, E, F, A, w, x, c, d, e, f, and a are as defined in thepreceding claims, and each S independently has no meaning or is H, OH,or a reactive moiety that allows for coupling the multiple releasespacer system to leaving groups Z, which may be the same or different,to afford compoundsV—(W—)_(w)(X—)_(x)C((A—)_(a)Z)_(c),V—(W—)w(X)_(x)C(D((A—)_(a)Z)_(d))_(c),V—(W—)_(w)(X—)_(x)C(D(E((A—)_(a)Z)_(e))_(d))_(c), andV—(W—)_(w)(X—)_(x)C(D(E(F((A—)_(a)Z)_(f))_(e))_(d))_(c), respectively.27. A compound according to claim 26 wherein the reactive moiety S isconnected via a carbonyl group to the multiple release spacer or spacersystem.
 28. A compound according to claim 27 wherein S representsN-succinimidyl-N-oxide, p-nitrophenoxide, pentafluorophenoxide, orchloride.
 29. A compound according to claim 26 wherein S is connected tothe methylene group of the multiple release spacer or spacer system. 30.A compound according to claim 29 wherein S represents chloride, bromide,p-toluenesulfonate, trifluoromethylsulfonate, or methylsulfonate.
 31. Acompound according to any of the preceding claims wherein the specifierV is a tripeptide.
 32. A compound according to claim 31 wherein thetripeptide is linked via its C-terminus to the self-eliminating multiplerelease spacer or spacer system.
 33. The compound of claim 32 whereinthe C-terminal amino acid residue of the tripeptide is selected fromarginine and lysine, the middle amino acid residue of the tripeptide isselected from alanine, valine, leucine, isoleucine, methionine,phenylalanine, cyclohexylglycine, tryptophan and proline, and theN-terminal amino acid residue of the tripeptide is selected from aD-amino acid residue and a protected L-amino acid residue includingprotected glycine.
 34. A compound according to claim 33 wherein thespecifier V is selected from D-alanylphenylalanyllysine,D-valylleucyllysine, D-alanylleucyllysine, D-valylphenylalanyllysine,D-valyltryptophanyllysine and D-alanyltryptophanyllysine.
 35. A compoundaccording to any of the preceding claims wherein the specifier V is anamino-terminal capped peptide covalently linked via the C-terminus tothe self-eliminating multiple release spacer or spacer system.
 36. Thecompound of claim 35 wherein the specifier V is selected frombenzyloxycarbonylphenylalanyllysine, benzyloxycarbonylvalyllysine,D-phenylalanylphenylalanyllysine, benzyloxycarbonylvalylcitrulline,tert-butyl oxycarbonylphenylalanyllysine,benzyloxycarbonylalanylarginylarginine,benzyloxycarbonylphenylalanyl-N-tosylarginine,2-aminoethylthiosuccinimidopropionylvalinylcitrulline,2-aminoethylthiosuccinimidopropionyllysylphenylalanyllysine,acetylphenylalanyllysine, andbenzyloxycarbonylphenylalanyl-O-benzoylthreonine.
 37. A compoundaccording to any of the preceding claims wherein the specifier Vcomprises a reactive moiety that can be used to couple said compound toa targeting moiety.
 38. A compound according to claim 37 in which thereactive moiety is

wherein X is een leaving group.
 39. A compound according to claim 37 inwhich the reactive moiety is an N-hydroxysuccinimide ester, ap-nitrophenyl ester, a pentafluorophenyl ester, an isothiocyanate, anisocyanate, an anhydride, an acid chloride, a sulfonyl chloride, and analdehyde.
 40. A compound according to claim 37 in which the reactivemoiety is a hydrazine group or an amino group.
 41. A compound accordingto any of the preceding claims wherein the specifier V comprises atargeting moiety.
 42. A compound according to claim 41 in which thetargeting moiety is selected from the group consisting of a protein orprotein fragment, an antibody or an antibody fragment, areceptor-binding or peptide vector moiety and a polymeric or dendriticmoiety.
 43. A compound according to any of the preceding claims selectedfrom the group consisting of

and salts thereof.
 44. The use of a compound according to claim 26-30 or37-40 for the preparation of a compound of claim
 4. 45. Diagnostic assayprocess in which a compound according to any of the preceding claims isused.
 46. Process according to claim 45 in which the presence or amountof an enzyme is determined.
 47. Process according to claim 46 in whichthe presence or amount of a protease is determined.
 48. Processaccording to claim 47 in which the compound that is used comprises asubstrate for said protease and leaving group Z is detected.
 49. Processaccording to claim 47 in which the compound that is used comprises asubstrate for an enzyme, which is the product of cleavage of itspro-enzyme precursor by said protease and leaving group Z is detected.50. A composite structure comprising two or more compounds according toany of the preceding claims, connected with a polymeric structure.
 51. Acompound according to any of the preceding claims, wherein the specifierV is removed or transformed by an enzyme that is transported to thevicinity of or inside target cells or target tissue via ADEPT, PDEPT,MDEPT, VDEPT, or GDEPT.
 52. Use of a compound according to any of thepreceding claims for the preparation of a pharmaceutical composition forthe treatment of a mammal being in need thereof.
 53. A pharmaceuticalcomposition comprising a compound according to any of claims 1 to 51.54. A process for preparing a pharmaceutical composition comprising thestep of mixing a compound according to any of claims 1 to 51 with apharmaceutically acceptable carrier.
 55. A method of treating a mammalbeing in need thereof, whereby the method comprises the administrationof a pharmaceutical composition according to claim 52 or 53 or isobtained according to the process of claim 54, to the mammal in atherapeutically effective dose.